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Glycosylation Methods and Analysis Sigma

Glycos

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0% found this document useful (0 votes)

598 views87 pages

Glycosylation Methods and Analysis Sigma

Glycos

Uploaded by

bogobon

We take content rights seriously. If you suspect this is your content, claim it here.

Available Formats

Download as PDF, TXT or read online on Scribd

Glycoprotein

Analysis

1st Edition

M A N U A L
Authors:
Robert Gates
Elner Rathbone
Lisa Masterson
Ian Wright
Asgar Electricwala

Glycan
Classification
Glycoprotein
Purification
Deglycosylation
Strategies
Detection
Glycan Analysis
Product Directory

[Link]

Glycoprotein Analysis
Application Note
PNGase F Enzyme:
Efficient N-Deglycosylation
in Glycoprotein Analysis
By Elner Rathbone, Asgar Electricwala, and Ian Wright
Sigma-Aldrich Corporation, Poole, England
Introduction
Glycosylation is one of the most common post-translational
modifications of proteins in eukaryotic cells. These glycoproteins are involved in a wide range of biological functions such as receptor binding, cell signaling, immune
recognition, inflammation, and pathogenicity. Mammalian
glycoproteins contain three major types of oligosaccharides (glycans): N-linked, O-linked, and glycosylphosphatidylinositol (GPI) lipid anchors. N-Linked glycans are attached
to the protein backbone via an amide bond to an asparagine
residue in an Asn-Xaa-Ser/Thr motif, where X can be any
amino acid, except Pro. O-Linked glycans are linked via the
hydroxyl group of serine or threonine. Variation in the
degrees of saturation at available glycosylation sites results
in heterogeneity in the mass and charge of glycoproteins.
To study the structure and function of a glycoprotein, it is
often desirable to remove all or just a select class of glycans.
This approach allows the assignment of specific biological
functions to particular components of the glycoprotein.
The removal of N-linked glycans from glycoproteins eliminates heterogeneity in matrix-assisted laser desorption/
ionization-time of flight (MALDI-TOF) MS analysis. Also,
removal of glycans may enhance or reduce the blood
clearance rate and/or potency of a glycoprotein therapeutic. Although sites of potential N-glycosylation can be
predicted from the consensus sequence Asn-Xaa-Ser/Thr,
it cannot be assumed that a site will actually be glycosylated. Therefore the sites of glycan attachment must be
identified and characterized by analytical procedures.
Peptide-N-glycosidase F (PNGase F) is one of the most widely
used enzymes for the deglycosylation of glycoproteins.
The enzyme releases asparagine-linked (N-linked) oligosaccharides from glycoproteins and glycopeptides. A tripeptide
with the glycan-linked asparagine as the central residue
is the minimum substrate for PNGase F. The glycan can
be a high-mannose, hybrid, or complex type. However,
N-glycans with fucose linked a(1,3) to the Asn-bound
N-acetylglucosamine are resistant to the action of PNGase F.
MALDI-TOF MS is a widely used technique for rapid identification of proteins separated by gel electrophoresis.
Glycopeptides, however, suffer from signal suppression

during MS analysis due to the heterogeneity of the

attached glycans. In this application note, we have used
PNGase F enzyme (Product Code P 7367) for the deglycosylation of a model glycoprotein, a1-antitrypsin, and have
identified and localized N-glycosylation sites by MALDI-MS
peptide mapping. The GlycoProfile I Enzymatic In-Gel
N-Deglycosylation Kit (Product Code PP 0200) conveniently
provides quality-tested reagents and a detailed protocol
for in-gel protein deglycosylation.
Efficient and convenient in-gel protein deglycosylation
A specific example demonstrating the use of the enzyme
is the deglycosylation of the glycoprotein a1-antitrypsin.
After reduction and alkylation (using tributylphosphine and
iodoacetamide contained in the ProteoPrep Reduction
and Alkylation Kit, Product Code PROT-RA), samples are
mixed with SDS-PAGE sample buffer and separated on
10% BisTris NuPAGE gel (Invitrogen Corp., Carlsbad, CA)
using MOPS running buffer. The gel is stained with 0.1%
Coomassie blue stain and destained.
The glycoprotein band from the 1D gel (or a spot from
a 2D gel) is carefully cut out and sliced into sections. The
pieces are destained, dried, and incubated with PNGase F.
The liquid surrounding the gel pieces is removed and
either discarded or retained for gylcan analysis if required.
The dried gel pieces are incubated with trypsin (Product
Code T 6567) and the surrounding liquid containing the
tryptic peptides is removed for further analysis.
Facile sample preparation for mass spectrometry
Sample preparation is simplified since the enzyme formulation does not contain detergents or stabilizers that
could interfere with analysis by MS. The solution of tryptic
peptides is concentrated and desalted on a C18 ZipTip
(Millipore Corporation, Billerica, MA) prior to analysis.
MS analysis was carried out on a MALDI-TOF instrument
fitted with a 337-nm UV laser. Peptides were analyzed
in positive ion reflectron mode using a-cyano-4-hydroxycinnaminic acid as the matrix. Spectra were acquired
over a mass range of 4000 m/z with matrix suppression
set at 800 mass units. Data analysis was carried out using
ProteinLynx software (Waters Corporation, Milford, MA).
Summary
PNGase F is an effective enzyme for the release of N-linked
glycans from glycoproteins, in gel or in solution. The formulation employed for PNGase F (Product Code P 7367)
provides a convenient, stable, freeze-dried product that
is compatible with MS analysis. This enzyme is also included
in the GlycoProfile In-Gel and GlycoProfile In-Solution
Deglycosylation Kits, which are composed of Proteomics
grade, high purity components for N-deglycosylation and
tryptic digestion.

Table of Contents
Overview
Key to Monosaccharide Symbols,
Abbreviations and Projections . . . . . . . . . . . . 02
Classification and Structure of
Glycan Components
N-Linked Glycans . . . . . . . . . . . . . . . . . . . 05
O-Linked Glycans . . . . . . . . . . . . . . . . . . . 09
GPI Anchored Proteins . . . . . . . . . . . . . . . 12
Glycoprotein Purification
m-Aminophenylboronate Matrices . . . . . . . . 13
Lectin Matrices . . . . . . . . . . . . . . . . . . . . . . . . 13
Glycoprotein Detection
Colorimetric Detection on PAGE and Blots .
Fluorescent Detection on PAGE . . . . . . . . .
Biotin Labeling on Blots . . . . . . . . . . . . . . .
Glycoprotein Standards . . . . . . . . . . . . . . .

.
.
.
.

15
16
17
18

Chemical Deglycosylation Strategies

Hydrazinolysis. . . . . . . . . . . . . . . . . . . . . . . . . 22
Alkaline b-Elimination . . . . . . . . . . . . . . . . . . 23
Trifluoromethanesulfonic Acid . . . . . . . . . . . . 23
Enzymatic Deglycosylation Strategies
N-Linked Glycan Enzymatic Hydrolysis .
Native and Sequential Hydrolysis . . . . .
O-Linked Glycan Enzymatic Hydrolysis .
Endoglycosidases . . . . . . . . . . . . . . . . .

.
.
.
.

24
25
29
32

Glycan Labeling and Analysis

Glycan Labeling . . . . . . . . . . .
HPLC Analysis . . . . . . . . . . . . .
Mass Spectrometry . . . . . . . . .
NMR Analysis . . . . . . . . . . . . .
Electrophoresis . . . . . . . . . . .

.
.
.
.
.

36
37
39
45
45

.
.
.
.
.

Glycan and Carbohydrate Standards

N-Linked High Mannose. . . . . . . . . . .
N-Linked Hybrid . . . . . . . . . . . . . . . . .
N-Linked Complex . . . . . . . . . . . . . . .
N-Linked Fragments . . . . . . . . . . . . . .
O-Linked Neutral Glycans . . . . . . . . . .
O-Linked Sialylated Glycans . . . . . . . .
Blood Group Antigens . . . . . . . . . . . .
Lewis and Cell Adhesion Glycans . . . .
Gal a(1,3) Gal Antigens . . . . . . . . . . .
Monosaccharides . . . . . . . . . . . . . . . .

.
.
.
.
.
.
.
.
.
.

47
49
49
55
57
60
63
65
67
67

Glycobiology Product Directory

Additional Information Sources:
Enzyme Explorer. . . . . . . . . . . . . . . . . .
Complete List of Glycolytic Enzymes . .
GPI Anchor Enzymes. . . . . . . . . . . . . . .
Glycosyltransferases . . . . . . . . . . . . . . .
Inhibitors of Carbohydrate Metabolism .
Glycolytic Enzyme Substrates . . . . . . . .
Neoglycoproteins . . . . . . . . . . . . . . . . .
Lectins. . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.
.
.

. 68
. 69
. 74
. 74
. 75
. 76
. 79
. 80

Overview
One of the distinguishing features of the
proteome in eukaryotic cells is that most
proteins are subject to post-translational
modification, of which glycosylation is the
most common form. It is estimated that
more than half of all proteins that have
been characterized are glycoproteins. The
carbohydrate components of glycoproteins
perform critical biological functions in
protein sorting, immune and receptor
recognition, inflammation, pathogenicity,
metastasis, and other cellular processes.
Mammalian glycoproteins contain three
major types of oligosaccharides (glycans):
N-linked, O-linked, and glycosylphosphatidylinositol (GPI) lipid anchors. N-Linked glycans
are linked to the protein backbone via an
amide bond to asparagine residues in an
Asn-X-Ser/Thr motif, where X can be any
amino acid, except Pro. O-Linked glycans
are linked to the hydroxyl group of serine

or threonine. GPI-anchored proteins are

attached at their carboxy-terminus through a
phosphodiester linkage of phosphoethanolamine to a trimannosyl glucosamine core
structure. The reducing end of the latter
moiety is bound to the hydrophobic region
of the membrane via a phosphatidylinositol group.
Variations in structure and degree of glycosylation site saturation can contribute to
overall mass heterogeneity. The terminal
residues of these glycans are commonly
N-acetylneuraminic acid (sialic acids). The
degree of sialylation affects both the mass
and charge of a glycoprotein. Other modifications to the protein such as sulfation
or phosphorylation also affect charge.
O-Linked glycans often have lower mass
than N-linked structures, but can be more
abundant and heterogeneous.

Key to Monosaccharide Symbols, Abbreviations, and Projections

Symbols, Structure Projections, and Abbreviations for Monosaccharides
Monosaccharide

3-D Chair projection

Haworth projection

CH2OH
O

HO
OH

Fischer projection

H
HO

Glc

CH2OH

b-D-Glucose (Glc)

OH
[Link]

CH2OH
HO

HO
HO

b-D-Mannose (Man)

Symbol

OH
OH

H
CH2OH

Man

Key to Monosaccharide Symbols, Abbreviations, and Projections

Symbols, Structure Projections, and Abbreviations for Monosaccharides
Monosaccharide

3-D Chair projection

Haworth projection

CH2OH

Symbol

H
O

Fischer projection

Gal

H
OH

CH2OH

b-D-Galactose (Gal)

CH2OH
O

HO
OH

NHAc

H
NHAc

GlcNAc

H
CH2OH

b-D-N-Acetylglucosamine (GlcNAc)

CH2OH

OH
HO

H
OH

OH
NHAc

NHAc

GalNAc

NHAc

b-D-N-Acetylgalactosamine
(GalNAc)

CH2OH
HO

OH
OH

b-D-Xylose (Xyl)

H
HO

OH
OH

Xyl

H
H

[Link]

O
HO

Key to Monosaccharide Symbols, Abbreviations, and Projections

Symbols, Structure Projections, and Abbreviations for Monosaccharides
Monosaccharide

3-D Chair projection

Haworth projection

Fischer projection

CO2H

HO
CH2OH
OH

CO2H

AcHN HO

AcHN

OH
OH
OH

CO2H

HO
OH
a-N-Acetylneuraminic acid
Sialic Acid (NeuNAc)

OH
CH2OH

CH2OH
CO2H

HO
O

OH
O

OH
OH

HO
OH

H
OH

H
CO2H
HO

O
OH
HO2C

IdoA

H
CO2H

OH
H3C
HO

[Link]

a-L-Iduronic acid (IdoA)

a-L-Fucose (Fuc)

CH2OH
OH

HO
HO

GlcA

b-D-Glucuronic acid (GlcA)

NeuNAc

AcHN

Symbol

CH3

O
OH

OH
OH

H
CH3

Fuc

Classification and Structure of Glycan Components

N-Linked Glycans
All eukaryotic cells express N-linked glycoproteins. Protein
glycosylation of N-linked glycans is actually a co-translational
event, occurring during protein synthesis. N-linked glycosylation requires the consensus sequence Asn-X-Ser/Thr.
Glycosylation occurs most often when this consensus
sequence occurs in a loop in the peptide. Oligosaccharide
intermediates destined for protein incorporation are synthesized by a series of transferases on the cytoplasmic
side of the endoplasmic recticulum (ER) while linked to
the dolichol lipid. Following the addition of a specific
number of mannose and glucose molecules, the orientation of the dolichol precursor and its attached glycan shifts
to the lumen of the ER where further enzymatic modification occurs. The completed oligosaccharide is then
transferred from the dolichol precursor to the Asn of the
target glycoprotein by oligosaccharyltransferase (OST).
Further processing includes trimming of residues such as
glucose and mannose, and addition of new residues via
transferases in the ER and, to a great extent, in the Golgi.
In the Golgi, high mannose N-glycans can be converted
to a variety of complex and hydrid forms which are unique
to vertibrates.

Inhibition or elimination of glycosylation in the study of

N-linked glycans can be brought about by a number of
compounds. In the presence of compactin, coenzyme Q,
and exogenous cholesterol, N-glycosylation is greatly
inhibited. Treatment with tunicamycin completely blocks
deglycosylation in that it inhibits GlcNAc C-1-phosphotransferase, which is critical in the formation of the dolichol
precursor necessary for synthesizing of N-glycans.
The diverse assortment of N-linked glycans are based on
the common core pentasaccharide, Man3GlcNAc2. Further
processing in the Golgi results in three main classes of
N-linked glycan sub-types; High-mannose, Hybrid, and
Complex. Complex glycans contain the common trimannosyl core. Additional monosaccharides may occur in
repeating lactosamine units. Additional modifications may
include a bisecting GlcNAc at the mannosyl core and/or
a fucosyl residue on the innermost GlcNAc. Complex
glycans exist in bi-, tri- and tetraantennary forms.

[Link]

Basic N-linked Structure

[Link]

High-Mannose Structure

[Link]

Hybrid Structure

[Link]

Complex Structure (tetraantennary)

O-Linked Glycans
O-Linked glycans are usually attached to the peptide chain
through serine or threonine residues. O-Linked glycosylation is a true post-translational event and does not require
a consensus sequence and no oligosaccharide precursor
is required for protein transfer. The most common type
of O-linked glycans contain an initial GalNAc residue (or
Tn epitope), these are commonly referred to as mucin-type
glycans. Other O-linked glycans include glucosamine, xylose,
galactose, fucose, or manose as the initial sugar bound to
the Ser/Thr residues. O-Linked glycoproteins are usually
large proteins (>200 kDa) that are commonly bianttennary
with comparatively less branching than N-glycans.
Glycosylation generally occurs in high-density clusters
and may contribute as much as 50-80% to the overall mass.
O-Linked glycans tend to be very heterogeneous, hence
they are generally classified by their core structure. Nonelongated O-GlcNAc groups have been recently shown to
be related to phosphorylation states and dynamic processing

related to cell signaling events in the cell. O-Linked glycans

are prevalent in most secretory cells and tissues. They
are present in high concentrations in the zona pelucida
surrounding mammalian eggs and may funtion as sperm
receptors (ZP3 glycoprotein). O-Linked glycans are also
involved in hematopoiesis, inflammation response mechanisms, and the formation of ABO blood antigens.
Elongation and termination of O-linked glycans is carried
out by several glycosyltransferases. One notable core
structure is the Gal-b(1-3)GalNAc (core 1) sequence that
has antigenic properties. Termination of O-linked glycans
usually includes Gal, GlcNAc, GalNAc, Fuc, or sialic acid.
By far the most common modification of the core
Gal-b(1-3)-GalNAc is mono-, di-, or trisialylation. A less
common, but widely distributed O-linked hexasaccharide
structure contains b(1-4)-linked Gal and b(1-6)-linked
GlcNAc as well as sialic acid.

[Link]

Di- and Trisialated O-Linked Core

O-Linked Core 2 Hexasaccharide

Core 1

Core 2

Core 3

[Link]

Core 4

Core 5

Core 6

Core 7

[Link]

Core 8

GPI Anchor Glycoproteins

Glycosylphosphatidylinisotol (GPI) anchored proteins are
membrane bound proteins found throughout the animal
kingdom. GPI anchored proteins are linked at their carboxyterminus through a phosphodiester linkage of phosphoethanolamine to a trimannosyl-non-acetylated glucosamine
(Man3-GlcN) core. The reducing end of GlcN is linked to
phosphatidylinositol (PI). PI is then anchored through
another phosphodiester linkage to the cell membrane
through its hydrophobic region. Intermediate forms are
also present in high concentrations in microsomal preparations. The Man3-GlcN oligosaccharide core may undergo
various modifications during secretion from the cell.

Their functionality ranges from enzymatic to antigenic

and adhesion. They contribute to the overall organization
of membrane bound proteins and are important in apical
protein postioning. GPI-anchored proteins also play a
critical role in a variety of receptor-mediated signal
transduction pathways.
Release of GPI anchored proteins can be accomplished by
treatment with Phospholipase C, Phosphatidylinositolspecific (PLC-PI) (Product Codes P 5542 and P 8804). The
enzyme specifically hydrolyzes the phosphodiester bond
of phosphatidylinositol to form a free 1,2-diacylglycerol
and glycopeptide-bound inositol cyclic-1,2-phosphate.

[Link]

GPI Anchor

Purification of proteins selectively utilizing their glycan

component as a capture target is commonly done utilizing affinity chromatography. The most popular affinity
matrices are m-aminophenylboronic acid agarose and
the immobilized lectins, Con A and Wheat Germ.

m-Aminophenylboronic Acid Matrices

m-Aminophenylboronic acid matrices are capable of
forming temporary bonds with any molecule that
contains a 1,2-cis-diol group.

Glycoprotein Purification

Glycoprotein Purification
Procedure
Equilibration buffers should be of low ionic strength,
with pH 7-9.
For a column volume of 1 ml, apply 1-2 mg of protein in
approximately 250 ml of buffer: 50 mM taurine/NaOH,
pH 8.7, containing 20 mM MgCl2.
Optimize the column flow rate to 2 ml/hour, collecting
2 ml fractions.
Elute the bound protein using the same buffer with
50 mM sorbitol or 50 mM Tris/HCl added.
References
1. Mallia, A.K. et al., Anal. Letters, 14 (B8), 649-661 (1981).
2. Immobilized Affinity Ligand Techniques, Hermanson, G.T., et al.,
Eds. (Academic Press, 1992), pp. 338, 339-392.
3. Affinity Chromatography: A Practical Approach, P.D.G Dean, W.S.
Johnson and F. A. Middle, Eds., (IRL Press, 1985), p. 133.

Aminophenylboronate Matrices
A 8530

m-Aminophenylboronic Acid
Affinity Medium

Matrix: cross-linked 6% beaded agarose

Activation: epoxy, with attachment through the amino group, with a 12-atom spacer
Ligand immobilized: 5-20 mmoles per ml
Form: (light pink) suspension in 0.5 M NaCl, with 0.1 M sodium acetate, pH 5.0
Synonym: PBA-agarose

A 4046

m-Aminophenylboronic Acid
Affinity Medium

Matrix: acrylic beads

Activation: oxirane, with attachment through the amino group, with a 5-atom spacer
Ligand immobilized: 300-600 mmoles per gram
Form: lyophilized powder
Swelling: 1 g swells to approximately 4 ml

A 8312

m-Aminophenylboronic Acid
Affinity Medium

Matrix: 6% beaded agarose

Activation: epichlorohydrin, with attachment through amino group with a 9-atom spacer
Ligand immobilized: 40-90 mmoles per ml
Binding Capacity: 8-14 mg Peroxidase Type VI per ml
Form: suspension in water containing 0.002% chlorhexidine diacetate.

(For additional Lectins see page 80)

Concanavalin A matrices bind specifically to mannosyl
and glucosyl residues of polysaccharides and glycoproteins. Unmodified hydroxyl groups at the C3, C4, and
C6 positions of D-glucopyranosyl or D-mannopyranosyl
rings may be essential for binding. Con A matrices have
been used with SDS (0.05%) and TRITON X-100.
Procedure
Equilibration and Binding:
Pre-wash column with 5 column volumes of wash
solution (1 M NaCl, 5 mM MgCl2, 5 mM MnCl2, and
5 mM CaCl2).
Equilibrate column in the buffer of choice (pH ranges
generally between 6.5 to 7.5 although buffers as low as
pH 4.1 and as high as 9.0 have been used successfully).
A commonly used starting buffer is 20 mM Tris, pH 7.4,
containing 0.5 M NaCl.
Load sample solution in equilibration buffer (protein
concentrations 1-20 mg/ml, free of particulates).
Wash the resin with equilibration buffer until eluent is
protein free.

Elution:
Elute the target protein with gradient or step-wise
elution with methyl a-D-glucopyranoside or methyl
a-D-mannopyranoside, glucose, or mannose (5 mM 500 mM).
Maximum recovery and cleaning of the resin may be
achieved by using 1 M sucrose, glucose, mannose, or
corresponding a-methyl glycoside. The addition of
chaotropic agents (0.5 M to 6 M) may also be required
for maximum recovery, but these denaturing conditions
may severely damage the resin. Therefore they should
only be used as a last resort.

[Link]

Lectin Matrices

Glycoprotein Purification

Glycoprotein Purification
Con A Matrices
C 9017

Concanavalin A Immobilized

Matrix: Sepharose 4B
Activation: cyanogen bromide
Ligand immobilized: 10-15 mg per ml
Binding Capacity: 20-45 mg thyroglobulin per ml
Form: suspension in 0.1 M acetate buffer, pH 6.0, containing 1 M NaCl, 1 mM each of
CaCl2, MgCl2, and MnCl2 and 0.01% thimerosal

C 6170

Concanavalin A Immobilized

Matrix: 4% beaded agarose

Activation: cyanogen bromide
Ligand immobilized: approx. 15 mg per ml
Binding Capacity: 6 mg yeast mannan per ml
Form: suspension in 0.1 M acetate buffer, pH 6.0, containing 1 M NaCl, 1 mM each of
CaCl2, MgCl2, and MnCl2 and 0.02% thimerosal

References
1. Yahara, I. and Edelman, G.M., Restriction of the mobility of lymphocyte immunoglobulin receptors by concanavalin A. Proc. Nat.
Acad. Sci. USA, 69(3), 608-612 (1972).
2. Li, Y., et al., The p185neu-containing Glycoprotein Complex of a
Microfilament-associated Signal Transduction Particle. J. Biol.
Chem., 274(36), 25651-25658 (1999).

3. Romero, P.A., et al., Glycoprotein biosynthesis in Saccharomyces

cerevisiae. Partial purification of the alpha-1,6-mannosyltransferase that initiates outer chain synthesis. Glycobiology, 4(2),
135-140 (1994).

Wheat Germ (Triticum vulgaris) matrices are specific for

GlcNAc2 or NeuNAc residues.

Elution:
Elute the target protein with gradient or step-wise
elution with equilibration buffer containing 100 mg/ml
N-acetylglucosamine (Product Code A 8625).

Procedure
Equilibration and Binding:
Wash and equillibrate column with 5 column volumes
of wash solution (0.05 M sodium phosphate, pH 7.0,
containing 0.2 M NaCl).
Load sample solution in equilibration buffer (protein
concentrations 1-20 mg/ml, free of particulates).
Wash the resin with equilibration buffer until eluent is
protein free.

WGA Matrices
L 1882

Wheat Germ (Triticum vulgaris)

Lectin Immobilized

Matrix: Cross-Linked 4% beaded agarose

Activation: cyanogen bromide
Ligand immobilized: 5-10 mg per ml
Form: Suspension in 1.0 M NaCl and 0.02% thimerosal

L 1394

Wheat Germ (Triticum vulgaris)

Lectin Immobilized

Matrix: 6% agarose macrobeads

Activation: cyanogen bromide
Ligand immobilized: Approx. 6 mg per ml
Binding Capacity: 1-2 mg ovomucoid per ml
Form: Suspension in 0.9% NaCl and 0.01% thimerosal

References

[Link]

1. Lotan, R., et al., Activities of lectins and their immobilized derivatives in detergent solutions. Implications on the use of lectin
affinity chromatography for the purification of membrane glycoproteins. Biochem., 16, 1787-1794 (1977).
2. Janicott, M., et al., The insulin-like growth factor 1 (IGF-1) receptor is responsible for mediating the effects of insulin, IGF-1, and
IGF-2 in Xenopus laevis oocytes. J. Biol. Chem., 266, 9382 (1991).

Glycoprotein Detection

Initial detection of glycoproteins in vitro is routinely accomplished on SDS-PAGE gels and

Western blots. Cellular localization of glycoproteins is normally accomplished utilizing
lectin fluorescent conjugates.
For a complete list of lectins including fluorescent labeled lectins, see page 80.

Glycoprotein Detection Kit

Product Code GLYCO-PRO
Product Description
The Glycoprotein Detection Kit provides a system to easily
detect the sugar moieties of glycoproteins on SDS-PAGE or
on Western blotting membranes. This detection system is
a modification of Periodic acid-Schiff (PAS) methods and
yields magenta bands with a light pink or colorless background. The detection limits have been found to be
25-100 ng of carbohydrates depending on the nature
and the degree of glycosylation of proteins. Peroxidase

from horseradish, reported as having a carbohydrate

content of approximately 16%, is used as a positive
control in the kit. The table below describes the steps
and time required when utilizing this kit.
Contains sufficient materials to stain 10 mini gels
(8 x 10 cm) or 5 large gels (16 x 18 cm) or same sizes
of blotting membranes.
Components
Oxidation Component (Periodic Acid)
Reduction Component (Sodium Metabisulfite)
Schiffs Reagent, Fuchsin-Sulfite Reagent
Peroxidase

Steps

Time for gel thickness 0.5-0.75 mm or for membrane

Time for gel thickness 1.0-1.5 mm

1. Fixing

30 min.

60 min.

2. Washing

2 x 10 min.

2 x 20 min.

3. Oxidation

30 min.

60 min.

4. Washing

2 x 10 min.

2 x 20 min.

5. Staining

1-2 hours or until bands turn magenta

6. Reduction

60 min.

120 min.

7. Washing

Band color will intensify with changes of fresh water

Band color will intensify with changes of

fresh water

8. Storage

overnight

[Link]

Colorimetric Detection on PAGE and

Western blots

Glycoprotein Detection
Fluorescent Detection on PAGE Gels
Glycoprotein Detection

GlycoProfile III
Fluorescent Glycoprotein Detection Kit
8 Product Code PP0300
Product Description
GlycoProfile III is designed for the fluorescent in-gel
detection of glycosylated proteins utilizing standard
UV-transillumination. Following SDS-PAGE, proteins are
fixed in the gel with an acetic acid/methanol solution.
The carbohydrates on the proteins are oxidized to aldehydes with periodic acid. A hydrazide dye is reacted with
the aldehydes, forming a stable fluorescent conjugate.
This allows for the specific, sensitive detection of the
glycoproteins directly in gels. This kit can also be used
to detect glycoproteins after Western transfer to PVDF
membranes.

Ovalbumin (glycosylated and phosphorylated)

b-Casein (not glycosylated, phosphorylated)
RNase B (glycosylated, not phosphorylated)

Rabbit

Mouse

Rabbit

Figure 1. PTM Marker (2 ml of a 6-fold dilution), containing glycosylated and non-glycosylated proteins, was separated by electrophoresis
on a 420% SDS-PAGE gel. The gel was stained for glycoproteins
with GlycoProfile III (left), imaged, and then stained for total protein
with EZBlue Gel Staining Reagent (right). The glycoproteins appear
as bright fluorescent bands. Each band represents approximately
300 ng of protein.
Mouse

The classical method for in-gel carbohydrate detection

uses Periodic Acid-Schiff reagent (PAS). It has a detection
limit of 25-100 ng of carbohydrate. PAS staining of glycoproteins is very selective, but lacks the sensitivity of fluorescent detection (5-25 ng of carbohydrate). The limit of
detection varies with the glycoprotein and the degree
and type of glycosylation. Typical detection limits observed
with the ProteoProfile PTM Marker (P 1745) are 150 ng
of ovalbumin (5 ng of carbohydrate) and 150 ng of RNase B
(30 ng carbohydrate). The detection limits are 5-10 times
lower than those observed with the Periodic Acid-Schiff
reagent.

BSA (not glycosylated, not phosphorylated)

Sufficient reagents are supplied for 10 mini gels.

Components

IgG Heavy Chain (glycosylated)

Oxidation Reagent
Glycoprotein Staining Reagent
Staining Buffer

IgG Light Chain

ProteoProfile PTM Marker

Designed as both a positive and negative control for SDS-PAGE gels
and Western blots of proteins with post-translation modifications.

[Link]

Specificity
Although this staining procedure is quite selective for
glycoproteins, some non-specific protein staining may
occur and may be more pronounced in some gel formulations. Staining the gel with EZBlue Gel Staining
Reagent after fluorescent imaging will allow identification of non-specifically stained proteins.
An alternative method is to run duplicate gels and fluorescently stain the second gel omitting the oxidation
step. Any fluorescent staining will be non-specific.

Figure 2. Mouse IgG and rabbit IgG were separated on a 420%

SDS-PAGE gel and stained in the same manner as the gel in Figure 1.
The IgG heavy chains, which are glycosylated, react strongly with
the fluorescent detection reagent. 2.5 mg of protein was applied
to each lane.

Glycoprotein Detection
colorimetric TMB or chemiluminescent CPS-1 peroxidase
substrate solutions. Alkaline phosphatase conjugated
streptavidin can also be used to probe the blots. Typical
detection limits for glycoproteins using this is method
are approximately 50-100 ng.

Biotin-hydrazide modification of periodate oxidized glycans can be utilized to label glycoproteins on Western
blots. The blots can then be probed with streptavidinperoxidase.1 Detection can be accomplished with either

Glycan

Optimal results are obtained on PDF membranes.

Biotin Labeled Glycan

Periodate
Oxidized
Glycan
O

Periodate

Glycoprotein Detection

Detection of Biotin Labeled

Glycoproteins on Western blots

Biotin Hydrazide

O=C

C=O

H2C

CH2 = CH2 = CH2 = CH2 = C = N = NH2

H2C
CH2

CH2
H2C

H2C

CH2
H
N

S
N
H

CH2

H
N

N
H

Detection Products
B 7639

Biotin Hydrazide

S 5512

Streptavidin, Peroxidase Labeled

S 1878

Sodium (meta) Periodate

T 0565

Tetramethylbenzidine Liquid Substrate System

CPS1-60

Chemiluminescent Peroxidase Substrate 60 ml

CPS1-120

Chemiluminescent Peroxidase Substrate 120 ml

CPS1-300

Chemiluminescent Peroxidase Substrate 300 ml

Reference

[Link]

1. Bayer, E.A., et al., Meth. Enzymol., 184, 415 (1990).

Glycoprotein Detection

Glycoproteins and
Glycoprotein Standards
There are many methods currently utilized in glycoprotein
detection and identification. Among the most common
are SDS-PAGE, Mass Spectroscopy, HPLC, NMR, and
Western blot. It is helpful and often necessary to include
glycoproteins as standards and/or molecular weight markers.

In biological assays involving cell or animal models, the

physiologic activity of glycoproteins is of primary interest.
Sigma-Aldrich offers a broad variety of glycoproteins for
analytical and biological use as standards, controls, and
bioactive components. The following products are an
abbreviated list of the most popular glycoproteins in
common analytical and biological assays. For additional
products please visit our website at [Link].

Preferred Glycoprotein Standards

I 0408
8

Invertase Glycoprotein Standard

Invertase is an enzyme that catalyzes the hydrolysis of sucrose into fructose and glucose.
Invertase Glycoprotein Standard is the periplasmic (glycosylated form, external invertase)
with 50% of its mass as polymannan. The periplasmic invertase molecule can exist in a
number of association states each a multiple of the core glycosylated monomer, a 60 kDa
peptide plus oligosaccharide chains and, depending on extraction, purification, and storage
conditions, will exist as a dimer, tetramer, hexamer, or octamer. Since yeast can provide an
alternative system for protein glycosylation that is similar to mammalian systems, periplasmic invertase is often used as a model for the study of the function of oligosaccharides in
glycoproteins and for studies on glycoprotein biosynthesis.

R 1153
8

RNase B Glycoprotein Standard

Bovine pancreatic Ribonuclease B (RNase B) is a glycoprotein that contains only N-linked

glycans. It is a globular protein composed of a single domain that occurs naturally as a
lesser component in a mixture along with Ribonuclease A (RNase A) which is the nonglycosylated core form. RNase B contains a single glycosylation site at Asn34 at which
from five to nine mannose residues are attached to the chitobiose core, i.e. Man5GlcNAc2,
Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2 and Man9GlcNAc2. Due to the heterogeneity
in the glycosylation at Asn34, RNase B exists as five glycosylated variants, with a molecular
weight of approximately 15,000 Da.

[Link]

Key Glycoproteins for Scientific Research

G 9885

a1-Acid Glycoprotein, human

99%, Purified from Cohn Fraction VI

A 5566

Monoclonal Anti-a1-Acid
Glycoprotein antibody

produced in mouse, Clone AGP-47, Ascites fluid, liquid

A 0534

Anti-a1-Acid Glycoprotein antibody

produced in rabbit, IgG fraction of antiserum, Lyophilized powder

A 3418

Anti-a1-Acid Glycoprotein antibody

produced in goat, Whole antiserum, liquid

A 9285

a1-Antichymotrypsin
from human plasma

approx. 95% (SDS-PAGE) Lyophilized powder

Glycoprotein is specific inhibitor of chymotrypsin-like serine proteases.
Contains Tris buffer salt and NaCl

A 9024

a1-Antitrypsin
from human plasma

Salt-free, lyophilized powder, a1-Proteinase inhibitor, serine protease inhibitor; inhibits trypsin,
chymotrypsin, pancreatic and granulocytic elastase, and acrosin. Effective concentration
equimolar with proteinase. Chromatographically prepared and partially purified.

G 0516

a2-hs-Glycoprotein
from human plasma

minimum 90% (SDS-PAGE) Lyophilized from 20 mM Tris-HCl, pH 8.0, with 200 mM NaCl

A 1960

Aggrecan
from bovine articular cartilage

Lyophilized powder, salt essentially free

sterile-filtered (dialyzed against water)

A 2512

Albumin
from chicken egg white
(Ovalbumin)

Grade VI approx. 99% (agarose gel electrophoresis) Lyophilized powder

Extent of labeling 5-6 mol mannose per mol ovalbumin protein

A 5503

Albumin
from chicken egg white
(Ovalbumin)

Grade V minimum 98% (agarose gel electrophoresis) Lyophilized powder

salt essentially free

A 5378

Albumin
from chicken egg white
(Ovalbumin)

Grade III minimum 90% (agarose gel electrophoresis) Lyophilized powder

A 4781

Asialofetuin
from fetal calf serum

Type I, salt essentially free

N-acetylneuraminic acid <0.5%

Glycoprotein Detection

Asialoglycophorin
from human blood Type MN

Lyophilized powder, Predominantly Asialoglycophorin A

G 7900

Monoclonal Anti-Glycophorin A
(a) antibody

Produced in mouse, Ascites fluid, Clone E4, Liquid

G 7650

Monoclonal Anti-Glycophorin A,B

(a,d) antibody

Produced in mouse, Clone E3, Ascites fluid, Liquid

G 7775

Monoclonal Anti-Glycophorin C
(b) antibody

Produced in mouse, Clone E5 or 2C10, Whole antiserum, Liquid

B 9277

Monoclonal Anti-Band 3 antibody

Produced in mouse, Clone BIII-136, Ascites fluid, Liquid

R 4011

Monoclonal Anti-Red Cell Wrb

Antigen antibody

Produced in mouse, Affinity purified, One unit will bind 1.0 mg of d-biotin
binds to Wrb, a composite blood group antigen resulting from the association between
Glycophorin A and Band 3

A 8706

Avidin
from egg white, recombinant

Affinity purified, One unit will bind 1.0 mg of d-biotin

B 8041

Biglycan
from bovine articular cartilage

Essentially salt-free, lyophilized powder, sterile-filtered

Interacts with collagen type I, as well as with fibronectin and TGF-b.

C 1063

Chorionic gonadotropin human

Lyophilized powder, sterile-filtered

vial of 2,500 I.U.

C 5297

Chorionic gonadotropin human

Lyophilized powder, Potency: approx. 3,000 I.U. per mg

G 4877

Gonadotropin
from pregnant mare serum

1,500-6,000 I.U./mg, PMSG

C 0755

Conalbumin chicken egg white

(Ovotransferrin)

Substantially iron-free, Minimum 98%

D 8428

Decorin
from bovine articular cartilage

Salt-free, lyophilized powder, sterile-filtered

Decorin interacts with collagen type I and II, fibronectin, thrombospondin, and TGF-b.

F 2379

Fetuin
from fetal calf serum

Lyophilized powder (from sodium acetate buffer)

free N-acetylneuraminic acid approx. 0.2%

F 3004

Fetuin
from fetal calf serum

Lyophilized powder
Further processing of F 2379 by gel filtration.

F 3879

Fibrinogen
from human plasma

Type I, Contains approx. 15% sodium citrate and approx. 25% sodium chloride.
Approx. 60% protein (80-90% of protein clottable)

F 4385

Fibrinogen
from murine plasma

Contains approx. 20% sodium citrate and approx. 30% sodium chloride.
Approx. 50% protein (over 80% of protein clottable)

F 8630

Fibrinogen
from bovine plasma

Type I-S powder, powder containing approx 75% protein, approx. 10% sodium citrate
and approx. 15% sodium chloride

F 4883

Fibrinogen
from human plasma

Essentially plasminogen-free powder containing approx. 50% protein, approx. 20% sodium
citrate and approx. 30% sodium chloride

H 7017

Hemocyanin Megathura crenulata

(keyhole limpet)

Lyophilized powder, contains stabilizing buffer, mol wt 8,000-9,000 kDa

vial of 20 mg KLH (in approximately 100 mg total weight)

H 8283

Hemocyanin Megathura crenulata

(keyhole limpet)

in PBS solution, Chromatographically purified

Concentration 3-7 mg/ml protein (A280)

K 3009

Keratan sulfate proteoglycan

from bovine cornea

sterile-filtered

L 0520

Lactoferrin
from human milk

approx. 90% (SDS-PAGE) Lyophilized powder

Chromatographically purified. Contains sodium chloride

L 2020

Laminin
from Engelbreth-Holm-Swarm
murine sarcoma

1 mg/ml in Tris buffered NaCl cell culture, tested

sterile-filtered

M 1778

Mucin
from porcine stomach

Type III partially purified powder, Bound sialic acids, approx. 1%

M 3895

Mucin
from bovine submaxillary glands

Type I-S Lyophilized powder, Neuraminidase substrate

Bound sialic acids approx. 12%

[Link]

A 9791

T 1408

holo-Transferrin bovine

approx. 98%, iron-saturated

T 4132

holo-Transferrin human

minimum 98%, iron-saturated

Chemical Deglycosylation Strategies

Analysis of the glycan structure of glycoproteins normally requires enzymatic or chemical
methods of deglycosylation.

[Link]

Chemical Deglycosylation
Strategies

Removal of carbohydrates from glycoproteins is useful for a number of reasons:

To simplify analysis of the peptide portion of the glycoprotein
To simplify the analysis of the glycan component
To remove heterogeneity in glycoproteins for X-ray crystallographic analysis
To remove carbohydrate epitopes from antigens
To enhance or reduce blood clearance rates of glycoprotein therapeutics
To investigate the role of carbohydrates in enzyme activity and solubility
To investigate ligand binding
For quality control of glycoprotein pharmaceuticals

Chemical Deglycosylation Strategies

Hydrazinolysis

Chemical Deglycosylation
Strategies

Hydrazine hydrolysis has been found to be effective in

the complete release of unreduced O- and N-linked
oligosaccharides. Selective and sequential release of
oligosaccharides can be accomplished by initial mild
hydrazinolysis of the O-linked oligosaccharides at 60 C
followed by N-linked oligosaccharides at 95 C. Hydrazine
hydrolysis leaves the glycan intact but results in destruction of the protein component.
Glycan release is accomplished by the addition of fresh
anhydrous hydrazine to a salt-free, freshly lyophilized
glycoprotein sample. The volume of hydrazine should
produce a protein concentration of 5 to 25 mg/ml. The
reaction mixture should be capped immediately. For the
release of both N- and O-linked glycans incubation should
be for 4 hours at 95 C. For the selective release of
O-linked glycans, incubation at 60 C for 5 hours is suitable.
Excess unreacted hydrazine can be removed under high
vacuum at a temperature not exceeding 25 C. The vacuum
pump should be equipped with an activated charcoal/
alumina trap. Addition of small aliquots of anhydrous
toluene may be required to bring the sample to complete dryness.
The dried sample should then be re-N-acetylated on ice
by the addition of ice-cold saturated sodium bicarbonate
solution, followed immediately by the addition of acetic
anhydride. The sample is mixed gently and incubated at
room temperature for 10 minutes. A second equal aliquot
of acetic anhydride is added and incubated for an additional
20 minutes. A 5-fold molar excess of acetic anhydride
over the amine content of the protein should be used.
The volume of sodium bicarbonate added should yield
a 0.5 M acetic anhydride final concentration in the reaction mixture.

A small amount of the released glycan pool may exist as

the acetohydrazide derivative. These derivatives may be
converted to the unreduced glycans by resuspension of
the dryed glycan pool in 1 mM Cu(II) acetate in 1 mM
acetic acid and incubation at room temperature for
one hour.
Dowex 50WX2 can be used to remove the cations and
the sample is washed through with water.
Glycan and protein components can be separated by gel
filtration or by paper chromatography.
Complete details for this procedure are given in Methods
in Enzymology. Care should be taken during all steps of
this procedure as many of the reagents and reactions are
extremely hazardous and highly reactive.
References
1. Patel, T., Bruce, J., Merry, A.H., Bigge, J.C., Wormald, M.R., and
Parekh, R.B., Biochemistry, 32, 679-693 (1992).
2. Takasaki, S., and Kobata, A., Meth. Enzymol., 50, 50-54 (1978).

Key Products
H 2761

Hydrazine

24,451-1

Toluene, Anhydrous

S 6297

Sodium Bicarbonate

A 6404

Acetic Anhydride

C 5893

Cupric Acetate

21,747-6

Dowex 50WX2-400

Peptide
CH2OH
R
O

CH2OH

H
N

O
HO

C
NH

H3C

CH2

O
CH
C

NH2
NH

Hydrazinolysis

C
H3C

Peptide

H
N

O
HO

C
O

[Link]

Re-N-acetylation
CH2OH
R
O

CH2OH
O

HO
NH
H3C

C
O

Regeneration of
reducing
terminus

O
HO

C
NH

NH
H3C

H
N

C
O

CH3

Alkaline b-Elimination

Trifluoromethanesulfonic Acid

Release of glycans by alkaline b-elimination utilizes ammonium hydroxide/carbonate or sodium hydroxide (in conjunction with sodium borohydride).

Trifluoromethanesulfonic acid (TFMS) hydrolysis leaves an

intact protein component, but results in destruction of
the glycan.

O-Glycosidic linkages between glycans and the b-hydroxyl

groups of serine or threonine are readily hydrolyzed by
dilute alkaline solutions (0.05 to 0.1 M sodium hydroxide
or potassium hydroxide) under mild conditions (45 to 60 C
for 8 to 16 hours) leading to the liberation of O-glycans
by the mechanism of b-elimination. To prevent isomerization or degradation of the carbohydrates by peeling
reactions, the hydrolysis is performed in the presence of
a reducing agent (0.8 to 2 M sodium borohydride). This
results in the formation of the reduced (alditol) forms of
the glycans. N-Linked glycans are not cleaved under these
conditions, and neither are O-glycans attached to tyrosine,
hydroxyproline, and hydroxylysine. Furthermore, the
b-elimination reaction does not take place if the glycan
is attached to serine or threonine at the carboxy-terminus
of the protein.

Glycoproteins from animals, plants, fungi, and bacteria

have been deglycosylated by this procedure. It has been
reported that the biological, immunological, and receptor
binding properties of some glycoproteins are retained
after deglycosylation by this procedure, although this
may not be true for all glycoproteins. The reaction is
non-specific, removing all types of glycans, regardless of
structure, although prolonged incubation is required for
complete removal of O-linked glycans. Also, the innermost Asn-linked GlcNAc residue of N-linked glycans
remains attached to the protein. The method removes
the N-glycans of plant glycoproteins that are usually
resistant to enzymatic hydrolysis.

For quantitative release of N-linked glycans, harsher

alkaline conditions are required (1 M sodium hydroxide
at 100 C for 6 to 12 hours). Again, the reaction must be
performed under reducing conditions (1 to 2 M sodium
borohydride) to prevent peeling reactions taking place
on the released N-glycans. N-Acetylglucosamine (GlcNAc)
residues are de-acetylated during this reaction and must
be re-N-acetylated (acetic anhydride in methanol) during
the recovery of the glycans.

Chemical Deglycosylation
Strategies

Chemical Deglycosylation Strategies

References
1. Patel, T.P., and Rarekh, R.B., Meth. Enzymol., 230, 57-66 (1994).
2. Bendiak, B. and Cumming, D.A., Carbohydr. Res., 151, 89 (1986).
3. Makino, Y., et al., J. Biochem., 128, 175-180 (2000).
4. Piller, F., and Piller, V., in Glycobiology: A Practical Approach,
Fukuda, M. and Kobata, A. (Eds), pp. 291-328 (IRL/Oxford Univ.
Press, (Oxford UK 1993)).
5. Burgess, A.J., and Norman, R.I., Eur. J. Biochem. 178, 527-533
(1988).
6. Edge, A.S.B., J. Biochem., 376, 339-350 (2003).
7. Sojar, H.T., and Bahl, O.P., Meth. Enzymol, 138, 41-350 (1987).
8. Edge, A.S.B., et al., Anal. Biochem., 118, 131-137 (1981).

References
1. Carlson, D.M., and Blackwell, C., J. Biol. Chem., 243, 616-626 (1968).
2. Lloyd, K.O., Burchell, J., Kudryashov, V., Yin, B.W.T., and TaylorPapadimitriou, J., J. Biol. Chem., 271, 33325-33334 (1996).
3. Morelle, W., Guyetant, R., and Strecker, G., Carbohydr. Res., 306,
435-443 (1998).

GlycoProfile IV Chemical
Deglycosylation Kit
Product Code PP0510

The GlycoProfile Chemical

Deglycosylation Kit has been optimized to provide a rapid, convenient, and reproducible method to
remove glycans from glycoproteins by
reaction with trifluoromethanesulfonic acid
(TFMS). The deglycosylated protein can then be recovered
using a suitable downstream processing method. The kit
contains sufficient reagents and a glycoprotein standard,
for a minimum of 10 reactions when the sample size is
between 1 and 2 mg of a typical glycoprotein. Unlike other
chemical deglycosylation methods, hydrolysis with anhydrous TFMS is very effective at removing O- and N-linked
glycans (except the innermost Asn-linked GlcNAc or
GalNAc) and results in minimal protein degradation.

Visit
[Link]/glycogo
to find out more about the latest
glycobiology products
from Sigma-Aldrich
[Link]

COMING
SOON!

Enzymatic Deglycosylation Strategies

Sequential hydrolysis of individual monosaccharides from glycans can be useful for the
elucidation of the structure and function of the glycan component. Due to the restraints
of the specificity of glycolytic enzymes currently available, sequential hydrolysis of individual
monosaccharides is also required in many instances in order to completely remove a glycan
component enzymatically. This is particularly true in the enzymatic deglycosylation of
many O-linked glycans.

N-Linked Glycan Strategies

Enzymatic Deglycosylation
Strategies

Use of the enzyme PNGase F is the most effective method

of removing virtually all N-linked oligosaccharides from
glycoproteins. The oligosaccharide is left intact and, therefore, suitable for further analysis (the asparagine residue
from which the sugar was removed is deaminated to
aspartic acid, the only modification to the protein). A
tripeptide with the oligosaccharide-linked asparagine as
the central residue is the minimal substrate for PNGase F.
However, oligosaccharides containing a fucose a(1-3)-linked
to the asparagine-linked N-acetylglucosamine, commonly
found in glycoproteins from plants or parasitic worms,
are resistant to PNGase F. N-Glycosidase A (PNGase A),
isolated from almond meal, must be used in this situation.
This enzyme, however, is ineffective when sialic acid is
present on the N-linked oligosaccharide. Other commonly

used endoglycosidases such as Endoglycosidase H and

the Endoglycosidase F series are not suitable for general
deglycosylation of N-linked sugars because of their limited
specificities and because they leave one N-acetylglucosamine
residue attached to the asparagine.
Steric hindrance slows or inhibits the action of PNGase F
on certain residues of glycoproteins. Denaturation of the
glycoprotein by heating with SDS and 2-mercaptoethanol
greatly increases the rate of deglycosylation.
Through sequential deglycosylation of monosaccharides,
all complex oligosaccharides can be reduced to the trimannosyldiacetylchitobiose core by selective hydrolysis with
a neuraminidase, b-galactosidase, and N-acetylglucosaminidase, available as part of the Enzymatic Deglycosylation
Kit (Product Code E-DEGLY). Fucosidases may be required
in some situations.

PNGase F

[Link]

R = N- and C-substitution by groups other than H

R = H or the rest of an oligosaccharide
R3 = H or a(1-6)fucose

Enzymatic Deglycosylation Strategies

Enzymatic Deglycosylation
Strategies

Triantennary Glycans

Enzymatic Deglycosylation Strategies

[Link]

Enzymatic Deglycosylation
Strategies

Complex Tetraantennary Glycans

Enzymatic Deglycosylation Strategies

There is no enzyme comparable to PNGase F for removing

intact O-linked sugars. Monosaccharides must be sequentially hydrolyzed by a series of exoglycosidases until only
the Gal-b(1-3)-GalNAc core remains. O-Glycosidase can
then remove the core structure intact with no modification of the serine or threonine residue. Denaturation of
the glycoprotein does not appear to significantly enhance
O-deglycosylation. Any modification of the core structure
can block the action of O-Glycosidase. The most common
modification of the core Gal-b(1-3)-GalNAc is a mono-, di-,
or tri-sialylation. These residues are easily removed by
a(2-3,6,8,9)-Neuraminidase since only this enzyme is capable of efficient cleavage of the NeuNAc-a(2-8)-NeuNAc
bond. Another commonly occurring O-linked hexasaccharide
structure contains b(1-4)-linked galactose and b(1-6)-linked
N-acetylglucosamine as well as sialic acid. Hydrolysis of

this glycan will require, in addition to neuraminidase, a

b(1-4)-specific galactosidase and an N-acetylglucosaminidase.
A non-specific galactosidase will hydrolyze b(1-3)-galactose
from the core glycan and leave O-linked GalNAc that cannot
be removed by O-Glycosidase. b(1-4)-Galactosidase and
b-N-acetylglucosaminidase can be used for the hydrolysis
of these and any other O-linked structures containing
b(1-4)-linked galactose or b-linked N-acetylglucosamine
such as polylactosamine. Less common modifications that
have been found on O-linked oligosaccharides include
a-linked galactose and a-linked fucose. Directly O-linked
N-acetylglucosamine (found on nuclear proteins) and
a-linked N-acetylgalactosamine (found in mucins) have
also been reported. Additional exoglycosidases are necessary for complete O-deglycosylation when these residues
are present. Fucose and mannose directly O-linked to
proteins cannot presently be removed enzymatically.

Enzymatic Deglycosylation
Strategies

O-Linked Glycan Strategies

Di- and Trisialated O-Linked Glycans

[Link]

O-Linked Core 2 Hexasaccharide

Enzymatic Deglycosylation Strategies

Deglycosylation Kits
GlycoProfile I
Enzymatic In-Gel N-Deglycosylation Kit

Enzymatic Deglycosylation
Strategies

Product Code PP0200

R1 = N- and C-substitution by groups other than H

R2 = H or the rest of an oligosaccharide structure
R3 = H or a(1-6)fucose

In-gel Deglycosylation

Excise protein
of interest
Place gel slice in
siliconized tube

Destain gel slice

Remove destaining
solution and dry
gel in Speed Vac

Rehydrate with
PNGase F solution

Incubate for
30 min. at 37 C

Add water

Incubate 2 hours to
overnight at 37 C
Remove glycancontaining supernatant.
Analyze if desired.

Glycosylation often leads to problems in subsequent protein analysis procedures. Glycopeptides generally do not
readily ionize during MS analysis leading to insufficient
spectral data. Furthermore, proteolytic digestion of the
native glycoprotein is often incomplete due to steric
hindrance by the oligosaccharides. Removal of the
carbohydrate groups from a glycoprotein prior to
protein identification is preferred.
Sigmas GlycoProfile I Enzymatic In-gel N-Deglycosylation
Kit is optimized to provide a convenient and reproducible
method to N-deglycosylate and tryptically digest protein
samples in 1D or 2D polyacrylamide gel slices for subsequent MS or HPLC analysis. The procedure is suitable for
Coomassie Blue and Colloidal Coomassie stained gels and
may be used with gels silver stained and destained using
Sigmas Proteo Silver Plus Kit (Product Code PROT-SIL2).
GlycoProfile Enzymatic In-gel N-Deglycosylation kit includes
the enzymes and reagents necessary for N-linked deglycosylation and tryptic digestion. The samples can then be
desalted and concentrated for analysis by MALDI-TOF or
electrospray MS.
Features & Benefits
Provides all components for in-gel deglycosylation and
trypsinization of protein samples Conveniently prepares deglycosylated protein samples for analysis by
MS or HPLC
Utilizes PNGase F for the enzymatic removal of N-linked
glycans Proteins remain intact, unlike the use of chemical deglycosylation which can degrade the protein
Includes Proteomics Grade PNGase F and Trypsin
Highly purified enzymes possess no unwanted activities
or additives to complicate analysis
PNGase F is supplied lyophilized from a low salt buffer
Allows reconstitution of the enzyme to any concentration needed
Works in solution or with gel slices Allows choice
of methods
Components
Destaining Solution
Proteomics Grade PNGase F

Wash gel slice

with water

[Link]

Remove and
discard liquid

Proteomics Grade Trypsin

Trypsin Solubilization Reagent
Trypsin Reaction Buffer
Invertase (positive control)
Peptide Extraction Solution

Dry gel slice in

Speed Vac

Sample is now ready to be

rehydrated in Trypsin solution
(see Trypsin In-Gel Digest kit).

Biotech Grade Acetonitrile

Enzymatic Deglycosylation Strategies

GlycoProfile II
Enzymatic In-Solution N-Deglycosylation Kit
The GlycoProfile Enzymatic In-Solution N-Deglycosylation
Kit has been optimized to provide a convenient and reproducible method to remove N-linked glycans from glycoproteins and is compatible with subsequent MALDI-TOF
mass spectrometric analysis without interference from
any of the reaction components. The kit contains sufficient enzyme, glycoprotein standard and reagents, for
a minimum of 20 reactions when the sample size is
between one to two mg of a typical glycoprotein.
Features & Benefits
Provides all components for in-solution N-linked deglycosylation of protein samples Conveniently prepares
deglycosylated protein samples for analysis by MS,
HPLC and PAGE
Reagents are optimized for direct MS analysis No need
for post-reaction sample clean up
Utilizes PNGase F for the enzymatic removal of N-linked
glycans Proteins remain intact, unlike the use of chemical deglycosylation which can degrade the protein
Includes Proteomics Grade PNGase F and Trypsin
Highly purified enzymes possess no unwanted activities
or additives to complicate analysis
PNGase F is supplied lyophilized from a low salt buffer
Allows reconstitution of the enzyme to any concentration needed
Components

Enzymatic Deglycosylation
Strategies

Product Code PP0201

R1 = N- and C-substitution by groups other than H

R2 = H or the rest of an oligosaccharide structure
R3 = H or a(1-6)fucose

In-solution Deglycosylation

Aliquot glycoprotein sample

Add denaturant

Incubate at 100 C for 10 min

Cool to room temperature

Proteomics Grade PNGase F

Ribonuclease B
10x Reaction Buffer

Add reaction buffer and PNGase F

Octyl b-D-Glucopyranoside
Incubate at 37 C for 1 hour

Analyze by MS, PAGE or HPLC

[Link]

2-Mercaptoethanol

Enzymatic Deglycosylation Strategies

Key Endoglycosidases
PNGase F
Synonyms: Glycopeptidase F; N-Glycosidase F; Peptide
N-Glycosidase F; Peptide-N4-(acetyl-b-glucosaminyl)asparagine amidase
EC [Link]

Molecular Weight: 36 kDa

Enzymatic Deglycosylation
Strategies

PNGase F cleaves all asparagine-linked complex, hybrid,

or high mannose oligosaccharides unless b(1-3) core
fucosylated. The asparagine must be peptide bonded
at both termini. The asparagine residue from which the
glycan is removed is deaminated to aspartic acid.
Detergent and heat denaturation increases the rate of
cleavage up to 100 times. Most native proteins can still
be completely N-deglycosylated, but incubation time must
be increased. The optimal pH is 8.6 and the enzyme is
active in the pH range of 6 to 10.

R1 = N- and C-substitution by groups other than H

R2 = H or the rest of an oligosaccharide structure
R3 = H or a(1-6)fucose

CH2OH
R
O

Peptide
CH2OH
R
O

O
HO

PNGase F

CH2
C

NH
H3C

H
N

H3C

Peptide

CH
C

O
NH

CH2

C
O

Peptide

Core GlcNAc

Asparagine Residue
Peptide

Aspartic Acid Residue

SDS-PAGE analysis of RNase B treated with varying amounts of PNGase F Enzyme

PNGase F Products
P 7367
8

PNGase F

Synonyms: N-Glycanase, N-Glycosidase F, Proteomics Grade

Features and Benefits
Excellent for applications requiring N-linked deglycosylation.
Superior performance in on-blot, in-gel, and in-solution digestion methods.
High activity, minimum 25,000 units per milligram.
Compatible for use in MALDI-TOF Mass Spectroscopy.
Each lot tested for suitability.

[Link]

Proteomics Grade PNGase F from Sigma-Aldrich is extensively purified and contains minimal
residual buffer salts and no glycerol or other stabilizers or preservatives that may interfere
in sensitive glycoprotein analysis methods.

Lane 1: Control (no PNGase F)

Lane 2-10: Test (+ 0.01 to 0.09 U PNGase F)

G 5166
8

PNGase F

Synonyms: N-Glycanase, N-Glycosidase F

from Chryseobacterium (Flavobacterium), Buffered Aqueous Solution

Enzymatic Deglycosylation Strategies

Deglycosylation Products
P 9120
8

PNGase F

Synonyms: N-Glycanase, N-Glycosidase F

Recombinant, Solution

N 3786

a(23,6,8,9) Neuraminidase

Synonyms: Sialidase, Acyl-neuraminyl Hydrolase, Proteomics Grade

from Arthrobacter ureafaciens

PP0200*
8

Glycoprofile I
In-Gel N-Deglycosylation Kit

Excellent performance when used for in-gel N-linked deglycosylation of glycoproteins

and glycopeptides. Utilizes extensively purified Proteomics Grade PNGase F and Trypsin.
Compatible with MS (TOF or Electrospray) analysis following desalting. Sufficient for a
minimum of 10 samples.

PP0201*
8

Glycoprofile II
In-Solution N-Deglycosylation Kit

Superior, reproducible method to remove N-linked glycans from glycoproteins. Utilizes

extensively purified Proteomics Grade PNGase F. Compatible with subsequent MALDI-TOF
mass spectrometric analysis, no need for desalting. Sufficient for a minimum of 20 reactions.

N-DEGLY

Native Deglycosylation Kit

The N-DEGLY kit includes Endoglycosidase F1, F2, and F3 as well as an optimized reaction
buffer for each. The kit is intended for use in the deglycosylation of N-linked oligosaccharides
from glycoproteins under native conditions. Particular residues, due to their location in
the native protein structure, are often resistant to the traditional deglycosylation methods
using PNGase F and cannot be removed unless the protein is denatured. Endoglycosidases
F1, F2, and F3 are less sensitive to protein conformation than PNGase F and are more suitable for deglycosylation of native proteins.

Enzymatic Deglycosylation
Strategies

Features and Benefits

Compatible for use in MALDI-TOP MS applications.
Cleaves all non-reducing, unbranched NANA/NeuNAc and NeuGc residues.
Ideal for complete, non-specific removal of sialic acid groups.

*For additional product information, refer to pages 30 and 31.

PNGase A
PNGase A (Glycopeptidase A, Peptide N-Glycosidase A),
hydrolyzes oligosaccharides containing a fucose a(1-3)linked to the asparagine-linked N-acetylglucosamine,
commonly found in glycoproteins from plants or parasitic
worms. These types of glycans are resistant to PNGase F.
Like PNGase F the asparagine asparagine residue from
which the glycan is removed is deaminated to aspartic
acid. However, it is ineffective when sialic acid is present
on the N-linked oligosaccharide.

R1 = N- and C-substitution by groups other than H

R2 = H or the rest of an oligosaccharide structure
R3 = H or a(1-6)fucose

G 0535

PNGase A

Buffered solution, min. 0.5 units/ml

G 1163

O-Glycosidase

O-Glycosidase hydrolyzes the serine- or threonine-linked unsubstituted O-Glycan core

[Gal-b(1-3)-GalNAc]. Any modification of the core structure can block the action of
O-Glycosidase.

E-DEGLY

Enzymatic Deglycosylation Kit

Completely removes all N- and simple O-linked carbohydrates from glycoproteins.

Efficiently digest polysialylated carbohydrates. Simplifies amino acid sequencing applications.
Removes carbohydrate epitopes from antigens. Native & denaturing procedures included
in bulletin.

[Link]

Deglycosylation Products

Enzymatic Deglycosylation Strategies

Endoglycosidase F1, F2 & F3

Enzymatic Deglycosylation
Strategies

Endoglycosidase F1 cleaves between the two N-acetylglucosamine residues in the N-linked diacetylchitobiose
glycan core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine
residue remaining on the asparagine. High mannose
(oligomannose) and hybrid structures can be removed
by Endoglycosidase F1, but not complex, oligosaccharides.
Useful under native or non-denaturing deglycosylation
conditions.

Endoglycosidase F3 cleaves between the two N-acetylglucosamine residues in the N-linked diacetylchitobiose
glycan core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine
residue remaining on the asparagine. Endo F3 cleaves
non-fucosylated biantennary and triantennary structures
at a slow rate, but only if peptide-linked. Core fucosylation of biantennary structures increases activity up to
400-fold. Endo F3 has no activity on oligomannose and
hybrid molecules. Endo F3 will also cleave fucosylated
trimannosyl core structures on free and protein-linked
oligosaccharides. Native deglycosylation of complex
tetrantennary glycans requires sequential hydrolysis
down to the trimannosyl-diacetylchitobiose core.

R1 = Asn or H
R2 = Oligomannose or hybrid configurations
R3 = H or a(1-6)fucose

Endoglycosidase F2 cleaves between the two N-acetylglucosamine residues in the N-linked diacetylchitobiose
glycan core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine
residue remaining on the asparagine. Endo F2 cleaves
biantennary complex and to a lesser extent high mannose oligosaccharides. Fucosylation has little effect on
Endo F2 cleavage of biantennary structures. Endo F2 will
not cleave hybrid structures. Useful under native or nondenaturing deglycosylation conditions.

R1 = N- and C-substitution by groups other than H

R2 = Biantennary and triantennary complex oligosaccharides or
trimannosylchitobiose core
R3 = H or a(1-6)fucose
R4 = Asn (H or Asn if core fucosylated)

R1 = H or Asn
R2 = Biantennary and oligomannose configurations
R3 = H or a(1-6)fucose

[Link]

Endoglycosidase F Products
E 9762

Endoglycosidase F1

Solution, from Chryseobacterium (Flavobacterium) meningosepticum

E 0639

Endoglycosidase F2

Solution, from Chryseobacterium (Flavobacterium) meningosepticum

E 2264

Endoglycosidase F3

Solution, from Chryseobacterium (Flavobacterium) meningosepticum

Enzymatic Deglycosylation Strategies

Endoglycosidase H cleaves between the N-acetylglucosamine residues of the chitobiose core of N-linked glycans,
leaving one N-acetylglucosamine residue attached to the
asparagine. The specificity of this enzyme is such that
oligomannose and most hybrid types of glycans, including

R1
R2
R3
R4

=
=
=
=

those that have a fucose residue attached to the core

structure, are cleaved whereas complex type glycans are
not released. Thus this enzyme is extremely useful for
selective release of oligomannose or hybrid type glycans
from glycoproteins. The enzyme is also active against
dolichol-linked glycans containing these structures.

Enzymatic Deglycosylation
Strategies

system, refractive index and pulsed amperometric detectors, with sensitivity in the
range of 10 to 100 pmol, have enabled
improved detection of native mono- and
oligosaccharides. Other approaches to
improve detection include labeling glycans
with radioactive isotopes, but this technique is not widely used due to the safety
requirements for the handling of radiolabeled substances.
Most glycoproteins exist as a heterogeneous
population of glycoforms or glycosylated
variants with a single protein backbone and
a heterogeneous population of glycans at

each glycosylation site. It has been reported

in the literature that for some glycoproteins,
100 or more glycoforms exist at each glycosylation site. In view of this heterogeneity
and the presence of branched structures,
the analysis of glycans is much more complicated than protein chemistry. It requires
several different strategies to separate and
study the structure of each individual glycan.
Once the glycans have been released from
the glycoprotein, the glycan pool can be
analyzed by MALDI-TOF mass spectrometry
or, after fluorescent labeling, by either HPLC
or MS, or both. This strategy can provide
a glycan profile or a glycosylation pattern that is highly characteristic of the
glycoprotein. The technology can be applied
to compare glycan profiles of glycoproteins
found in normal and diseased states, or to
compare different batches of recombinant
protein products. Both these techniques
provide valuable information in terms of
composition, linkage and arm specificity
(using various exoglycosidases) from which
structural information on individual glycans
can be elucidated.

Glycan Labeling
Chemical derivatization is now the most common method
used for labeling glycans at their reducing ends by reductive amination. A single molecule of fluorescent label can
be incorporated to each mono- or oligosaccharide, thus
allowing molar quantities to be determined. The sensitivity
Technique
HPLC

[Link]

Gel Electrophoresis

of detection by this technique is in the low femtomole

range and depending upon the analytical technique, an
appropriate fluorescent label can be used. The table below
lists examples of some of the fluorophores widely used
in glycan analysis.

Fluorescent Label

Abbreviation

Product Code

2-Aminobenzoic acid

2-AA

10678

2-Aminobenzamide

2-AB

10710

2-Aminopyridine

2-AP

09340

8-Aminonaphthalene-1,3,6-trisulfonic acid

ANTS

08658

2-Aminoacridone

AMAC

06627

Capillary Electrophoresis

9-Aminopyrene-1,3,6-trisulfonic acid

APTS

09341

Mass spectrometry

2-Aminobenzamide

2-AB

10710

Glycan Analysis and Labeling

HPLC Analysis of Glycans

Fetuin

16,000
12,000
8,000
4,000
0
70

100

110

120

130

Retention time (minutes)

Figure 2. HPLC Profile of the 2-AB labeled N-linked glycan library
obtained from Fetuin.

Partially hydrolyzed dextran, as a source of glucose oligomers, is used as an external standard and the elution
position of each peak is expressed in glucose units (gu)
(see Fig. 3). The elution positions of peaks in an unknown
glycan pool are assigned an overall gu value by comparison with the standard dextran ladder. These values may
then be used to predict possible structures for unknown
glycans. For example, it has been reported that the glycans
released from RNase B consist solely of N-linked oligomannose structures. The five peaks obtained upon profiling
of the glycans from RNase B can therefore be assigned
structures ranging from Man-5 to Man-9, as indicated
on Figure 1.
1

4
5

4,400

800

Glycan Analysis
and Labeling

Normal phase chromatography

The column used in this mode of chromatography consists
of a matrix with imide or amide functional groups. Samples
are applied in an organic solvent solution to the column
are then eluted with increasing concentration of aqueous
buffer. This exploits the subtle differences in hydrophilicity
between individual glycans and their affinity for the
column matrix, thereby achieving high resolution and
reproducibility. The ability to analyze sialylated and neutral
sugars in one chromatographic run makes it a useful technique for profiling both N- and O-glycan pools, and for
making comparisons between different glycan samples.
Figures 1 and 2 show the elution profiles of N-linked
glycans released from two model glycoproteins, RNase B
and Fetuin, respectively.

20,000

Fluorescence

The released glycan pool from glycoprotein hydrolysis

can be separated into components depending upon the
characteristics of the glycans and the HPLC matrices used.
Three types of chromatography, normal phase, weak anion
exchange, and reversed phase HPLC, have been generally
used with fluorescent-labeled oligosaccharides for the
separation of sialylated and neutral N- and O-linked
glycans. High pH anion exchange chromatography with
pulsed amperometric detector can be used with native
glycans, but has a major drawback in that the separation
is achieved in high molarity sodium hydroxide that has
to be removed to recover glycans for further structural
analysis.

RNase B
Fluorescence

Man-5

Fluorescence

600

Man-6

400

3,300

7
2,200

8
9
10
11

1,100

Man-8

200
Man-7

Man-9

0
70

100

110

120

Retention time (minutes)

100

120

140

160

Retention time (minutes)

Figure 3. Separation of partially hydrolyzed 2-AB labeled dextran on
normal phase HPLC. The numbers indicate glucose units (gu).

[Link]

Figure 1. HPLC Profile of the 2-AB labeled N-linked glycan library

obtained from RNase B.

Glycan Analysis and Labeling

Glycan Analysis
and Labeling

In some of these charge classes, for example in the

disialylated glycan peak area shown in Figure 4(A), the
partially-resolved peak contains bi- and triantennary
structures with the triantennary glycans eluting first.
The peaks corresponding to each charge band can be
collected, concentrated, further separated, and analyzed
by normal phase chromatography. Two examples are
shown in Figures 4(B) and 4(C).
A)

Fetuin glycan
library

Fluorescence

8,000

6,000

Neutral
Di

Tetra

Tri
4,000
Mono
2,000

0
0

Retention time (minutes)

B)
400

Neutral glycans

Fluorescence

300

200

100

[Link]

0
60

100

110

120

Retention time (minutes)

130

140

C)
400

Monosialylated glycans
300

Fluorescence

Weak anion exchange chromatography

Weak anion-exchange chromatography relies upon the
relative binding affinities of the glycans to the weak anion
exchange matrix in the column. This type of chromatography separates N- and O-linked glycans on the basis of
the number of charge groups they contain. The elution
position is therefore determined by the number of sialic
acid, sulfate, phosphate, or uronic acid moieties present
on the glycan, but also to some extent by the size of the
glycan. Thus, within one charge band, larger structures
elute before smaller ones. For example, Figure 4(A) shows
the separation of the N-linked glycan library released
from fetuin into five major peaks. The flow-through peak
contains neutral glycans, while peaks eluting with increasing time represent the mono-, di-, tri- and tetrasialylated
glycans of fetuin.

200

100

0
60

100

110

120

130

140

Retention time (minutes)

Figure 4. Separation of neutral and acidic glycans of fetuin by weak
anion-exchange chromatography. (A) The separation of the glycan
pool into neutral, mono-, di-, tri- and tetrasialylated glycans.
(B) Further separation of the neutral glycan fraction from (A) by
normal phase chromatography. (C) Further separation of the monosialylated glycan fraction from (A) by normal phase chromatography.

Reversed phase chromatography

Reversed phase chromatography separates sugars on the
basis of hydrophobicity and can be carried out on a C18
column. This method is complementary to the normal phase
chromatography, allowing some glycans that co-elute
on one system to be resolved by the other system. This
approach is important during structure determination of
the glycan to assess the shift in the retention time upon
treatment with a particular exoglycosidase. The sample is
applied in aqueous buffer to the column and eluted with
increasing concentration of organic solvent. Unlike the
normal phase chromatography, the elution positions of
glycans are measured by comparison with an arabinose
ladder and assigned as arabinose units (au).

Glycan Analysis and Labeling

MS of neutral glycans
Unlike peptides, neutral glycans exhibit low ionization
efficiency and the [M+H]+ ion is therefore not sufficiently
abundant. However, these glycans can be detected as
alkali metal adducts which ionize efficiently. Normally,
[M+Na]+ is the major ion, accompanied by a weaker [M+K]+
ion. Other adducts, such as [M+Li]+ can be generated by
the addition of the appropriate inorganic salt to the matrix.
The inclusion of NaCl in the matrix solvent allows the
glycans to ionize predominantly in the [M+Na]+ form
with little or no [M+K]+ ion formation.

Mass Spectrometry of Glycans

The development of modern mass spectrometry (MS)
equipment with high resolution and mass accuracy has
led to its use in analyzing glycans for both profiling and
structural studies. Unlike HPLC methods, a substantially
larger amount, about 10-20 times, of glycan is normally
required for a single MS spectrum. Matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) mass
spectrometry is the most widely used MS technique. The
information gained on using MALDI MS is mass weight
and this can be used to assign putative monosaccharide
structures present in a pure oligosaccharide since the mass
of a monosaccharide is measured with a high degree of
accuracy. Like HPLC, MALDI MS can also be used in conjunction with exoglycosidase enzymes for structural and
linkage analysis of glycans.
Different approaches and matrices are used for the analysis of neutral and acidic glycans (those that contain sialic
acid) by MALDI MS. Neutral glycans ionize with reasonable
efficiency using 2,5-dihydroxybenzoic acid (2,5-DHB)
(Product Code D 1942) as the matrix in positive ion mode.
Acidic glycans generally provide poor MALDI spectra with
DHB matrices due to variable losses of sialic acids or carboxyl groups leading to multiple peaks. These glycans
are therefore analyzed in negative ion mode using an
alternative matrix.

Glycan Analysis
and Labeling

Normally, use of the 2,5-DHB matrix is sufficient for most

applications. However, it has been reported that, in some
instances, a 2-3 fold increase in sensitivity can be obtained
with the use of Super 2,5-DHB matrix as it allows for
softer desorption with a reduction in metastable ion
formation. Super 2,5-DHB consists of a mixture (90:10
ratio, wt %) of 2,5-DHB and 2-hydroxy-5-methoxybenzoic
acid, respectively.
Ovalbumin (0.5 mg) was incubated with 7 units of PNGase F
enzyme (Product Code P 7367) at 37 C for 3 hours and
the glycans recovered by ultrafiltration. A third of this
material was dried under vacuum centrifugation and
then analyzed by MALDI-TOF MS. Figure 5 shows the
N-linked glycan profile of ovalbumin.

C
1136.22
100

A
933.20

D
1257.29

H
1745.52

E
1339.30
1152.26

974.16
%

G
1663.48

1298.30

F
1542.41
1419.33
1704.52
1501.41

975.16

J
I
1948.57
1866.59

B
1095.08
990.12

1762.53

991.14
1035.10

1964.66

K
2110.68

L
2151.74
m/z

900

1000

1100

1200

1300

1400

1500

Figure 5. Positive ion MALDI MS of the N-linked glycans from ovalbumin released by treatment with PNGase F. Recorded from 2,5-DHB

1600

1700

1800

1900

2000

2100

2200

2300

as the matrix. The letters indicate identified peaks, given on the

next pages.
[Link]

0
800

1907.65

1558.40

Glycan Analysis and Labeling

[Link]

Glycan Analysis
and Labeling

Corresponding Glycan Structures for MS Profile

Refer to Structure Key on pages 2-4

Glycan Analysis and Labeling

Refer to Structure Key on pages 2-4

[Link]

Glycan Analysis
and Labeling

Corresponding Glycan Structures for MS Profile

Glycan Analysis and Labeling

Glycan Analysis: General MS Protocol
4. Allow to dry at room temperature.
Note: Mass spectra can be acquired at this stage.
However the following steps have been reported to
improve signals by forming an even film of crystals,
which reduces the need to search for sweet spots
on the target.
5. Onto the dried spot, dispense 1 ml of ethanol and
allow the matrix plus analyte mixture to recrystallize
on the target plate.
6. Dry the spot. The target is now ready to acquire
mass spectra.
7. Figure 6 shows representative MALDI-TOF MS traces
obtained for three glycan standards Man-3 glycan,
Man-8 glycan, and NA4 glycan.

Procedure
The following method, commonly referred to as the
dried-droplet method, is based on the original MALDI
experiments and remains the most commonly used
method in the mass spectrometry community.
1. Transfer 3 ml of the appropriate matrix solution
(either DHB or Super DHB at 10 mg/ml) into a 0.5 ml
Eppendorf tube.
2. Add 1 ml of 10 pmol/ml solution of a standard glycan or
an unknown sample solution to the tube containing
the matrix. Vortex to mix.
3. Dispense an aliquot (approximately 1.5 ml) of this
mixture onto the MALDI target plate.
A)
933.32

Glycan Analysis
and Labeling

100

934.32

935.32

936.33
915.16

949.29

937.33

931.11

950.29

m/z
915

920

925

930

935

940

945

950

955

B)
1743.58

100

1744.59

1745.59

%
1746.59

1747.59
1760.56

[Link]

1748.59
0

m/z
1715

1720

1725

1730

1735

1740

1745

1750

1755

1760

1765

1770

1775

Glycan Analysis and Labeling

C)
2394.85

100
2393.85

2395.85

2396.86
%
2397.86
2398.86

2409.82

m/z
2360

2370

2380

2390

2400

2410

2420

2430

2440

Glycan Analysis
and Labeling

2350

Figure 6. MALDI-TOF mass spectra of (A) Man-3 glycan, (B) Man-8 glycan, and (C) NA4 glycan obtained in positive ion reflectron mode using
DHB as matrix. Each of these glycans are detected as the sodium adduct of the molecular ion [M+Na]+.

GlycoMass I Neutral Glycan

Calibration Kit
Product Code PP0301

COMING
SOON!

This kit provides a range of purified

N-linked glycans for the purpose of
testing MALDI mass spectrometers,
regardless of the instrument manufacturer. In addition to the glycans,
two different, high purity, re-crystallized
matrices and a ready-to-use matrix solvent are provided.
This kit provides the necessary reagents and standards for
the analysis of pure glycans, or for profiling and/or structure determination of complex glycan mixtures by MS.

Visit
[Link]/glycogo
to find out more about the latest
glycobiology products
from Sigma-Aldrich

GlycoMass I Neutral Glycan Calibration Kit is supplied

with the following items.

Monoisotopic [M]+ (Da) Monoisotopic [M+Na]+ (Da)

Amount Supplied

M 8418

Man-3 Glycan

910.3278

933.3175

1 nmol (approx.)

M 5918

Man-8 Glycan

1720.5919

1743.5817

1 nmol (approx.)

M 8918

NA4 Glycan

2370.8566

2393.8463

1 nmol (approx.)

D 1942

2,5-DHB matrix

2 x 10 mg

D 1817

Super 2,5-DHB matrix

2 x 10 mg

G 2164

Matrix solvent
(10% ethanol + 10 mM NaCl)

10 ml

[Link]

Product Code Product

Glycan Analysis and Labeling

MS of acidic glycans
DHB is the most widely used matrix for the MALDI MS
analysis of glycans. This matrix, however, is not ideal for
acidic glycans (such as sialylated N-linked sugars) as the
detection limit is poor compared to neutral glycans, and
fragmentation can occur with losses of carboxylic and/or
sialic acid groups. In addition, sialylated glycans analyzed
in the positive-ion mode yield a mixture of cation adducts
producing multiple peaks. One approach to enhance sensitivity is to derivatize the glycan, but this may compromise
the absolute quantitation of the glycan. An alternative
way is to choose a different matrix that can overcome the
problem of fragmentation during analysis and provide
an increased detection limit.

Glycan Analysis
and Labeling

Two most widely used matrices for the analysis of acidic

glycans are 6-aza-2-thiothymine and 2,4,6-trihydroxyacetophenone (THAP, Product Code 91928). The former
gives a significant increase in sensitivity over DHB matrix,
but still causes some fragmentation in the negative ion

linear mode. With THAP, the detection limit of analysis

can be at the 10 fmol level, with little or no evidence
of fragmentation in the linear mode. The conditions for
sample preparation with this matrix (outlined below) are
crucial for obtaining maximum sensitivity.
In order to prevent acid-catalyzed lactonization of sialic
acids, glycans were dissolved in 0.2 M ammonium hydroxide
solution before being applied to the MALDI target plate
and air dried. The spot was rehydrated with water and a
mixture of THAP and ammonium citrate (1 mg THAP in
1 ml of a 1:1 mixture of acetonitrile and 20 mM ammonium citrate) was applied. Vacuum drying of the sample
spot was used to prevent large crystals from forming. The
sample was then allowed to absorb moisture from the
atmosphere to promote the formation of small crystals.
Figure 7 is representative of a MALDI-TOF MS spectrum
obtained in negative ion linear mode for the mono-sialylated N-linked glycan A1.
1932.69

100

831.06
945.43
896.98

1075.39

1299.76

1525.73

1653.00

1997.02
2072.01

2169.84

2313.50

2613.30

0
800

m/z

900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900

Figure 7. MALDI-TOF mass spectrum of A1 glycan obtained in negative ion linear mode using THAP as matrix and detected as the [M-H] ion.

[Link]

References
1. Stahl, B., Steup, M., Karas, M., and Hillenkamp, F., Anal. Biochem.,
63, 1463-1466 (1991).
2. Karas, M., Ehring, H., Nordhoff, E., Stahl, B., Strupat, K., Hillenkamp,
F., Grehl, M., and Krebs, B., Org. Mass. Spectrom., 28, 1476-1481 (1993).
3. Harvey, D.H., Rapid Commun. Mass Spectrom., 7, 614-619 (1993).

Glycan Analysis and Labeling

NMR Analysis of Glycans
Nuclear magnetic resonance (NMR) analysis is based on
the extent that a glycan distorts a magnetic field. This
technique is non-destructive and can provide full structural
information. Hence this is the first analysis technique that
can be used on a purified glycan. However, a major limitation of this technique is that a relatively large amount, in
the range 10-100 ng, of purified glycan is required. With
higher MHz spectrometers and use of nano-probes, an
increased sensitivity can be achieved. Although this is a
powerful method, the high capital cost of equipment and
the requirements of personnel trained in its use tend
to limit its application in the field of glycoprotein and
glycan analysis.

Electrophoresis Products
08658

8-Aminonapthalene 1,3,6-trisulfonic acid sodium

salt (ANTS)

06627

2-Aminoacridone (AMAC)

14,644-7

7-Amino-1,3-napthalenedisulfonic acid (ANDS)

15,615-9

Sodium cyanoborohydride

D 8779

Dimethylsulfoxide (DMSO)

A 4058

40% Acrylamide solution

M 7279

N,N-Methylenebisacrylamide (BIS)

T 4904

10x Tris Glycine buffer, pH 8.3

A 3678

Ammonium sulfate

T 8133

N,N,NN-Tetramethylenediamine (TEMED)

Electrophoresis of Glycans

Figure 8. Electrofluorogram of ANTS-labeled N-linked glycans.

Electrophoresis was carried out in 20% resolving polyacrylamide gel
for 1 hr at 250V. Lanes 1, 2, 3 and 4 represent Man-3, Man-7, Man-8
and NA4 glycans, respectively.

Glycan Analysis
and Labeling

Glycan Sequencing
Enzymatic analysis of oligosaccharides using highly specific
exoglycosidases, either sequentially or in a matrix array,
is a powerful technique to determine the sequence and
structure of glycan chains. Initially, the glycan pool can
be separated into individual oligosaccharides and each
of these purified glycans can be digested sequentially with
various linkage-specific exoglycosidase enzymes. Figure 9
shows some of the most commonly used exoglycosidase
enzymes used to determine the structures of N-linked
glycans. After appropriate incubation, the product of
digestion can be analyzed by normal phase HPLC or by
MALDI MS. On the HPLC system, structures are assigned to
each peak from the known specificity of the exoglycosidase
used in the digestion and the pre-determined incremental
gu values of individual monosaccharide residues.
Sequencing of oligosaccharides present in a glycan pool
can also be carried out simultaneously on aliquots of the
pool using enzyme arrays. Products of several digestions
are resolved on normal phase HPLC in separate runs and
the shift in the retention times of individual peaks, due
to the action of single enzyme, can be noted. As each
individual exoglycosidase removes additional monosaccharides from the glycan chain, the successive shift in the
retention times, and hence their corresponding gu values,
are obtained. By comparison with the experimentally
determined incremental gu values for the addition of
a particular sugar residue to the standard glycan core
structure, possible structure to each peak present in the
original glycan pool can be assigned. These structural
assignments can further be confirmed by tandem MSn
mass spectrometry.

[Link]

Separation of glycans by electrophoresis in polyacrylamide

gel has been widely used and different methods are
described in the literature for analysis of monosaccharides
and oligosaccharides. The most commonly used system
is the electrophoresis of fluorophore-labeled glycans in
highly cross-linked polyacrylamide gels and is termed as
Fluorophore-Assisted Carbohydrate Electrophoresis (FACE).
The glycans are usually labeled with a fluorescent tag,
mainly ANTS or AMAC and separated on 20-40% gels.
The extent of cross-linking means that extra precautions
should be taken to prevent heating and warping of the
gel during the run. After electrophresis, the band patterns
are visualized by illuminating the gel under UV light and
photographing the image. Although this technique is sensitive in the sub-picomolar range, the resolution between
the glycans can be poor due to the limitation on the size
of the gel. The following figure shows the mobility of
four individual N-linked glycans after labeling with ANTS.
This method can also be used to obtain profiles of glycans
released from glycoproteins and to elucidate the structure of individual glycans after exoglycosidase digestion,
based on the mobility shift of the parent glycan band.

Glycan Analysis
and Labeling

Glycan Analysis and Labeling

Figure 9. Some of the exoglycosidase enzymes commonly used to determine the structure of N-linked glycans.

Refer to Structure Key on pages 2-4

[Link]

Exoglycosidases
A 6805

b-N-Acetylhexosaminidase

Recombinant, from Streptococcus pneumoniae, expressed in E. coli

F 8899

a-Fucosidase

From almond meal

F 5884

a-Fucosidase

From bovine kidney

G 4142

b-Galactosidase

From bovine testes

G 0413

b-Galactosidase

From Streptococcus pneumoniae

M 7257

a-Mannosidase

From Jack bean (Canavalia ensiformis)

N 3786

a-2-(3,6,8,9)-Neuraminidase

Proteomics grade, syn: a-sialidase, from Arthrobacter ureafaciens

N 7271

a-2,3-Neuraminidase

Recombinant, from Streptococcus pneumoniae, expressed in E. coli

Glycan and Carbohydrate Standards

M 8418

Man-3 Glycan

Syn: (Man)3(GlcNAc)2, Mannotriose-di-(N-acetyl-D-glucosamine), Oligomannose-3 glycan

70858-45-6, C34H58N2O26

M 2796

Man-3-F Glycan

Syn: M3F, Man3(Fuc)GlcNAc2, Mannotriose-[fucosyl-di-(N-acetyl-D-glucosamine)],

Oligomannose-3 glycan, core fucosylated
110387-51-4, C40H68N2O30

M 8912

Man-5 Glycan

Syn: (Man)5(GlcNAc)2, Mannopentaose-di-(N-acetyl-D-glucosamine), Oligomannose-5 glycan

66091-47-2, C46H78N2O36

Glycan and
Carbohydrate Standards

N-Linked High Mannose

[Link]

Refer to Structure Key on pages 2-4

Glycan and Carbohydrate Standards

N-Linked High Mannose
Man-5-Asn Glycan

Syn: (Man)5(GlcNAc)2Asn, Mannopentaose-di-(N-acetyl-D-glucosamine)-asparagine,

Oligomannose-5-Asn glycan
38784-68-8, C50H84N4O38

M 0420

Man-7 D1 Glycan

Syn: Man7(GlcNAc)2, Mannoheptaose-di-(N-acetyl-D-glucosamine) D1,

Oligomannose-7-D1 glycan
83178-05-06, C58H98N2O46

M 5918

Man-8 Glycan

Syn: Man8(GlcNAc)2, Mannooctaose-di-(N-acetyl-D-glucosamine) D1,D3,

Oligomannose-8 glycan
77036-51-2, C64H108N2O51

[Link]

Glycan and
Carbohydrate Standards

M 4164

Refer to Structure Key on pages 2-4

Glycan and Carbohydrate Standards

N-Linked High Mannose
M 9037

Man-9 Glycan

Syn: (Man)9(GlcNAc)2, Mannononaose-di-(N-acetyl-D-glucosamine)

71246-55-4, C70H118N2O56

M 9919

HYBR glycan

Syn: GlcNAc(GlcNAc)Man5(GlcNAc)2, HYBR with bisecting GlcNAc, Mannopentaose-di(N-acetylglucosamine), N-acetylglucosaminyl-bisecting-N-acetylglucosaminyl

84182-21-8, C62H104N4O46

Glycan and
Carbohydrate Standards

N-Linked Hybrid

N-Linked Complex
A1 Glycan

Refer to Structure Key on pages 2-4

Syn: NeuNAc(Gal-GlcNAc)2Man3(GlcNAc)2, Mannotriose-di-(N-acetylglucosamine),

sialyl-bis(galactosyl-N-acetylglucosaminyl), Monosialylated, galactosylated,
biantennary glycan
145211-79-6, C73H121N5O54

[Link]

M 2425

Glycan and Carbohydrate Standards

N-Linked Complex
A1F Glycan

Syn: NeuNAc(Gal-GlcNAc)2Man3(GlcNAc)2Fuc, Mannotriose-(fucosyl-di-[N-acetylglucosamine]),

mono-sialyl-bis(galactosyl-N-acetylglucosaminyl), Monosialyl galactosyl biantennary glycan,
core fucosylated
571188-30-2, C79H131N5O8

M 5800

A2 Glycan

Mannotriose-di-(N-acetyl-D-glucosamine), bis([N-accetyl-D-neuraminyl]-galactosyl[N-acetyl-D-glucosaminyl]) ammonium salt, Disialyl galactosyl biantennary glycan

71496-55-4, C84H138N6O62

M 2800

A2F Glycan

Syn: (NeuNAc-Gal-GlcNAc)2Man2(Fuc)(GlcNAc)2, Mannotriose-(fucosyl-di[N-acetylglucosamine]), bis(sialyl-galactosyl-N-acetylglucosaminyl), Disialylated,

galactosylated, fucosylated biantennary glycan, Disialyl galactosyl biantennary glycan,
core fucosylated
108341-22-6, C90H148N6O66

[Link]

Glycan and
Carbohydrate Standards

M 3800

Refer to Structure Key on pages 2-4

Glycan and Carbohydrate Standards

NA2 Glycan

Syn: (Gal-GlcNAc)2Man3(GlcNAc)2, Mannotriose-di-(N-acetyl-D-glucosamine),

bis(galactosyl-[N-acetyl-D-glucosaminyl]), Asialo galactosyl biantennary glycan
71496-53-2, C62H104N4O46

M 2300

NA2B Glycan

Syn: (Gal-GlcNac)2(GlcNAc)Man3(GlcNAc)2, Mannotriose-di-(N-acetylglucosamine),

bis(galactosyl-N-acetylglucosaminyl)-bisecting N-acetylglucosaminyl, Asialo, galactosylated,
biantennary glycan, bisecting GlcNAc, Asialo galactosyl biantennary glycan, with
bisecting GlcNAc
84632-71-3, C70H117N5O51

M 9419

NA2F Glycan

Syn: (Gal)2(GlcNAc)2(Man)3(GlcNAc)2Fuc, Mannotriose-[fucosyl-di-(N-acetylglucosamine)],

bis(galactosyl-N-acetylglucosaminyl), Asialo, galactosylated, biantennary glycan, core
fucosylated, Asialo galactosyl biantennary glycan, core fucosylated
142561-43-1, C68H114N4O50

Refer to Structure Key on pages 2-4

[Link]

M 5925

Glycan and
Carbohydrate Standards

N-Linked Complex

Glycan and Carbohydrate Standards

N-Linked Complex
NGA2 Glycan

Syn: (GlcNAc)2Man3(GlcNAc)2, Mannotriose-di-(N-acetylglucosamine),

bis(N-acetylglucosaminyl), Asialo, agalacto, biantennary glycan, Asialo agalacto
biantennary glycan
84808-02-6, C50H84N4O36

M 1925

NGA2F Glycan

Syn: (GlcNAc)2Man3(Fuc)(GlcNAc)2, Mannotriose-[fucosyl-di-(N-acetylglucosamine)],

bis(N-acetylglucosaminyl), Asialo, agalacto, biantennary glycan, core fucosylated,
Asialo agalacto biantennary glycan, core fucosylated
84825-26-3, C56H94N4O40

M 1800

NGA2FB Glycan

Syn: (GlcNAc)Man3(Fuc)(GlcNAc)2, Mannotriose-[fucosyl-di-(N-acetylglucosamine)],

bis(N-acetylglucosaminyl)-bisecting N-acetylglucosaminyl), Asialo, agalacto, biantennary
glycan, bisecting GlcNAc, core fucosylated, Asialo agalacto biantennary glycan, core
fucosylated, with bisecting GlcNAc
79726-49-1, C64H107N5O45

[Link]

Glycan and
Carbohydrate Standards

M 1675

Refer to Structure Key on pages 2-4

Glycan and Carbohydrate Standards

M 2925

A3 Glycan

Syn: (NeuNAc-Gal-GlcNAc)3Man3(GlcNAc)2, Mannotriose-di-(N-acetyl-D-glucosamine),

tris(sialyl-galactosyl-N-acetyl-D-glucosaminyl, Trisialylated, galactosylated, triantennary glycan,
Trisialyl galactosyl triantennary glycan
145164-24-5, C109H178N8O80

M 8793

NA3 Glycan

Syn: (Gal-GlcNAc)3-Man3-GlcNAc2, Mannotriose-di-(N-acetyl-D-glucosamine),

tris(galactosyl-N-acetyl-D-glucosaminyl, Asialo, galactosylated, triantennary glycan,
Asialo galactosyl triantennary glycan
82867-73-0, C76H127N5O56

Glycan and
Carbohydrate Standards

N-Linked Complex

[Link]

Refer to Structure Key on pages 2-4

Glycan and Carbohydrate Standards

N-Linked Complex
NGA3 Glycan

Syn: (GlcNAc)3Man3GlcNAc2, Mannotriose-di-(N-acetyl-D-glucosamine),

tris(N-acetyl-D-glucosaminyl), Asialo, agalacto, triantennary glycan,
Asialo agalacto triantennary glycan
110387-63-8, C58H97N5O41

M 8918

NA4 Glycan

Syn: [Gal-GlcNAc]4-Man3-GlcNAc2, Mannotriose-di-(N-acetylglucosamine), tetrakis

(galactosyl-N-acetyl-D-glucosaminyl), Asialo, tetraantennary N-linked glycan Asialo
galactosyl tetraantennary glycan
82867-74-1, C90H150N6O66

Glycan and
Carbohydrate Standards

M 2175

[Link]

Refer to Structure Key on pages 2-4

Glycan and Carbohydrate Standards

N-Asn

Syn: 2-Acetamido-1-b-(L-aspartamido)-1,2-dideoxy-D-glucose, b-D-GlcNAc-1N-Asn,

2-Acetamido-1-N-(b-L-aspartyl)-2-deoxy-b-D-glucopyranosylamine
2776-93-4, C12H21N3O8

A 3432

N2-Asn

Syn: N,N-Diacetyl-1-(N-b-L-aspartyl)-b-chitobiose, b-D-GlcNAc-(14)-b-D-GlcNAc-1-N-Asn,

2-Acetamido-4-O-(2-acetamido-2-deoxy-b-D-glucopyranosyl)-1-N-(b-L-aspartyl)-2-deoxy-bD-glucopyranosylamine
29625-73-8, C20H34N4O13

A 2292

MN Glycan

Syn: b-D-Man-(14)-D-GlcNAc, 2-Acetamido-2-deoxy-4-O-(b-D-mannopyranosyl)-Dglucopyranose

186765-90-2, C14H25NO11

A 6587

NM Glycan

Syn: b-D-GlcNAc-(12)-D-Man, 2-O-(2-Acetamido-2-deoxy-b-D-glucopyranosyl)-D-mannose

34621-73-3, C14H25NO11

A 5065

3a-Fucosyl-N-acetylglucosamine

Syn: a-L-Fuc-(13)-D-GlcNAc, 2-Acetamido-2-deoxy-3-O-a-L-fucopyranosyl-D-glucopyranose

24876-86-6, C14H25NO10

A 9566

4a-Fucosyl-N-acetylglucosamine

Syn: a-L-Fuc-(14)-D-GlcNAc, 2-Acetamido-2-deoxy-4-O-a-L-fucopyranosyl-D-glucopyranose

76211-71-7, C14H25NO10

M 1050

2a-Mannobiose

Syn: a-D-Man-(12)-D-Man, 2-O-a-D-Mannopyranosyl-D-mannopyranose

15548-39-7, C12H22O11

Refer to Structure Key on pages 2-4

[Link]

A 6681

Glycan and
Carbohydrate Standards

N-Linked Fragments

Glycan and Carbohydrate Standards

Glycan and
Carbohydrate Standards

N-Linked Fragments
M 6155

4a-Mannobiose

Syn: a-D-Man(14)-D-Man, 4-O-a-D-Mannopyranosyl-D-mannopyranose

35438-40-5, C12H22O11

M 7788

6a-Mannobiose

Syn: a-D-Man-(16)-D-Man, 6-O-a-D-Mannopyranosyl-D-mannopyranose

6614-35-3, C12H22O11

D 5422

3a,6a-Mannotriose

Syn: a-D-Man-(13)-[a-D-Man-(16)]-D-Man, 3,6-Di-O-(a-D-mannopyranosyl)-Dmannopyranose

121123-33-9, C18H32O16

M 0925

3a,6a-Mannopentaose

Syn: a-Man-(13)(a-Man-[16])-a-Man-(16)(a-Man-[13])-Man
112828-69-0, C30H52O26

[Link]

Refer to Structure Key on pages 2-4

Glycan and Carbohydrate Standards

N-Acetyllactosamine

Syn: N-Acetyl-4-O-(b-D-galactopyranosyl)-D-glucosamine, b-D-Gal-(14)-D-GlcNAc,

2-Acetamido-2-deoxy-4-O-b-D-galactopyranosyl-D-glucopyranose, N-acetyl-D-lactosamine
32181-59-2, C14H25NO11

A 6919

6b-GlcNAc-D-galactose

Syn: b-D-GlcNAc-(16)-D-Gal, 6-O-(2-Acetamido-2-deoxy-b-D-glucopyranosyl)-Dgalactopyranose, b6-GlcNAc-(16)-D-galactose

20212-77-5, C14H25NO11

A 8297

6b-GlcNAc-lactose

Syn: b-D-GlcNAc-(16)-b-D-Gal-(14)-D-Glc, 6-N-Acetylglucosaminyllactose,

4-O-(6-O-[2-Acetamido-2-deoxy-b-D-glucopyranosyl]-b-D-galactopyranosyl)-D-glucopyranose
68665-69-0, C20H35NO16

F 0393

2-Fucosyllactose

Syn: a-L-Fuc-(12)-b-D-Gal-(14)-D-Glc, 2-FL, 2-fucosyl-D-lactose

41263-94-9, C18H32O15

F 9641

3-Fucosyllactose

Syn: a-L-Fuc-(13)-[b-D-Gal-(14)]-D-Glc, 3-FL

41312-47-4, C18H32O15

L 6770

Lacto-N-tetraose

Syn: b-D-Gal-(13)-b-D-GlcNAc-(13)-b-D-Gal-(14)-D-Glc, LNT

14116-68-8, C26H45NO21

L 8526

Lacto-N-neo-tetraose

Syn: b-D-Gal-(14)-b-D-GlcNAc-(13)-b-D-Gal-(14)-D-Glc, LNnT

13007-32-4, C26H45NO21

Refer to Structure Key on pages 2-4

[Link]

A 7791

Glycan and
Carbohydrate Standards

O-Linked Neutral Glycans

Glycan and Carbohydrate Standards

[Link]

Glycan and
Carbohydrate Standards

O-Linked Neutral Glycans

L 8651

LDFT Glycan

Syn: 3,2-Difucosyllactose, a-L-Fuc-(12)-b-D-Gal-(14)-[a-L-Fuc-(13)]-D-Glc,

Lactodifucotetraose
20768-11-0, C24H42O19

L 5908

Lacto-N-fucopentaose I

Syn: a-L-Fuc (12)-b-D-Gal-(13)-b-D-GlcNAc-(13)-b-D-Gal-(14)-D-Glc, LNFP I

7578-25-8, C32H55NO25

L 6401

Lacto-N-fucopentaose II

Syn: Lea-Lactose, Lewis-a pentasaccharide, b-D-Gal-(13)-[a-L-Fuc-(14)]-b-D-GlcNAc(13)-b-D-Gal-(14)-D-Glc, LNFP II

21973-23-9, C32H55NO25

L 7777

Lacto-N-fucopentaose III

Syn: Lex-lactose, Lewis-X pentasaccharide, b-D-Gal-(14)-[a-L-Fuc-(13)]-b-D-GlcNAc(13)-b-D-Gal-(14)-D-Glc, LNFP III

25541-09-7, C32H55NO25

L 7902

Lacto-N-fucopentaose V

Syn: b-D-Gal-(13)-b-D-GlcNAc-(13)-b-D-Gal-(14)-[a-L-Fuc-(13)]-D-Glc, LNFP V

60254-64-0, C32H55NO25

Refer to Structure Key on pages 2-4

Glycan and Carbohydrate Standards

Lacto-N-difucohexaose I

Syn: Leb-lactose, Lewis-b hexasaccharide, a-L-Fuc-(12)-b-D-Gal-(13)[a-L-Fuc-(14)]b-D-GlcNAc-(13)-b-D-Gal-(14)-D-Glc, LNDFH I

16789-38-1, C38H65NO29

L 6645

Lacto-N-difucohexaose II

Syn: b-D-Gal-(13)-[a-L-Fuc-(14)]-b-D-GlcNAc-(13)-b-D-Gal-(14)-[a-L-Fuc-(13)]D-Glc, LNDFH II

62258-12-2, C38H65NO29

L 6654

Lacto-N-hexaose

Syn: b-D-Gal-(13)-b-D-GlcNAc-(13)-(b-D-Gal-[14]-b-D-GlcNAc-[16])-b-D-Gal(14)-D-Glc, LNH

64003-51-6, C40H68N2O31

L 8283

para-Lacto-N-hexaose

Syn: b-D-Gal-(13)-b-D-GlcNAc-(13)-b-D-Gal-(14)-b-D-GlcNAc-(13)-b-D-Gal-(14)D-Glc, pLNH

64331-48-2, C40H68N2O31

F 5171

Fucosyl-para-lacto-N-hexaose IV

Syn: b-Gal-(13)-b-GlcNAc-(13)-b-Gal-(1-4)(a-Fuc-[13])-b-GlcNAc-(13)-b-Gal-(14)Glc, FpLNH IV

115236-58-3, C46H78N2O35

Refer to Structure Key on pages 2-4

[Link]

L 7033

Glycan and
Carbohydrate Standards

O-Linked Neutral Glycans

Glycan and Carbohydrate Standards

[Link]

Glycan and
Carbohydrate Standards

O-Linked Sialylated Glycans

A 1314

3-Sialyl-3-fucosyllactose

Syn: a-NeuNAc-(23)-b-D-Gal-(14)-(a-Fuc-[13])-Glc, SFL,

3-N-acetylneuraminyl-3-fucosyllactose
122560-33-2, C29H48NNaO23

A 4564

LS-Tetrasaccharide a

Syn: LST a, a-NeuNAc-(23)-b-Gal-(13)-b-GlcNAc-(13]-b-Gal-(14)-Glc,

N-Acetylneuraminyllacto-N-tetraose a
64003-53-8, C37H62N2O29

A 4689

LS-Tetrasaccharide b

Syn: LST b, a-NeuNAc-(26)-(b-D-Gal-[13])-b-D-GlcNAc-(13)-b-D-Gal-(14)-Glc,

N-Acetylneuraminyllacto-N-tetraose b
64003-54-9, C37H62N2O29

A 4814

LS-Tetrasaccharide c

Syn: LST c, a-NeuNAc-(26)-b-Gal-(14)-b-GlcNAc-(13)-b-Gal-(14)-Glc,

N-Acetylneuraminyllacto-N-neo-tetraose c
64003-55-0, C37H61N2NaO29

Refer to Structure Key on pages 2-4

Glycan and Carbohydrate Standards

D 9045

Disialyllacto-N-tetraose

Syn: DSLNT, a-NeuNAc-(23)-b-Gal-(13)-[a-NeuNAc-(26)]-b-GlcNAc-(13)-b-Gal-(14)-Glc,

a-Neu5Ac-(23)-b-Gal-(13)-[a-Neu5Ac-(26)]-b-GlcNAc-(13)-b-Gal-(14)-Glc
61278-38-4, C48H77N3Na2O37

A 8681

3-Sialyllactose
from bovine colostrum
3-Sialyllactose
from human milk

Syn: NANA-Lactose, 3-N-Acetylneuraminyllactose, a-NeuNAc-(23)-b-D-Gal-(14)-D-Glc,

N-Acetylneuraminyllactose, 3-SL, a-Neu5Ac-(23)-b-D-Gal-(14)-D-Glc
35890-38-1, C23H39NO19

6-Sialyllactose
from human milk
6-Sialyllactose
from bovine colostrum

Syn: a-NeuNAc-(26)-b-D-Gal-(14)-D-Glc, 6-N-Acetylneuraminyllactose, 6-SL,

a-Neu5Ac-(26)-b-D-Gal-(14)-D-Glc
74609-39-5, C23H38NNaO19

A 9079

A 9204
A 8556

Glycan and
Carbohydrate Standards

O-Linked Sialylated Glycans

[Link]

Refer to Structure Key on pages 2-4

Glycan and Carbohydrate Standards

Glycan and
Carbohydrate Standards

O-Linked Sialylated Glycans

A 6936

3-Sialyl-N-acetyllactosamine

Syn: a-NeuNAc-(23)-b-D-Gal-(14)-D-GlcNAc, 3-N-Acetylneuraminyl-N-acetyllactosamine,

3-SLN
81693-22-3, C25H42N2O19

A 8431

6-Sialyl-N-acetyllactosamine

Syn: a-NeuNAc-(26)-b-D-Gal-(14)-D-GlcNAc, 6-N-Acetylneuraminyl-N-acetyllactosamine,

6-SLN, a-Neu5Ac-(26)-b-D-Gal-(14)-D-GlcNAc
136098-09-4, C25H41N2NaO19

A 0828

Sialyllactose
from human milk

Syn: N-Acetylneuraminyllactose, a-NeuNAc-(26)- and -(23)-b-D-Gal-(14)-D-Glc,

Neuramin-lactose, a-Neu5Ac-(26)- and -(26)-b-D-Gal-(14)-D-Glc
C23H38NNaO19

A 3307

Sialyllactose
from bovine colostrum

Syn: N-Acetylneuraminyl-lactose, a-NeuNAc-(23)- and -(26)-b-D-Gal-(14)-D-Glc,

Neuramin-lactose, a-Neu5Ac-(23)- and -(26)-b-D-Gal-(14)-D-Glc
C23H39NO19

[Link]

Refer to Structure Key on pages 2-4

Glycan and Carbohydrate Standards

4a-Galactobiose

Syn: a-D-Gal-(14)-D-Gal, 4-O-a-D-Galactopyranosyl-D-galactopyranose

80446-85-1, C12H22O11

G 9662

4b-Galactobiose

Syn: b-D-Gal-(14)-D-Gal, 4-O-b-D-Galactopyranosyl-D-galactopyranose

2152-98-9, C12H22O11

A 0167

Galacto-N-biose

Syn: b-D-Gal-(13)-D-GalNAc, 2-Acetamido-2-deoxy-3-O-b-D-galactopyranosylD-galactopyranose, T-Antigen

20972-29-6, C14H25NO11

A 9150

Lacto-N-biose

Syn: b-D-Gal-(13)-D-GlcNAc, 2-Acetamido-2-deoxy-3-O-(b-D-galactopyranosyl)D-glucopyranose

50787-09-2, C14H25NO11

F 7012

H-Disaccharide

Syn: a-L-Fuc-(12)-D-Gal, 2-O-a-L-Fucosyl-D-galactose

24656-24-4, C12H22O10

G 8394

4b-Galactosyllactose

Syn: b-D-Gal-(14)-b-D-Gal-(14)-D-Glc, 4-O-(4-O-b-D-Galactopyranosylb-D-galactopyranosyl)-D-glucopyranose

118396-93-3, C18H32O16

G 9287

Globotriose

Syn: a-D-Gal-(14)-b-D-Gal-(14)-D-Glc, 4-O-(4-O-a-D-Galactopyranosylb-D-galactopyranosyl)-D-glucopyranose

66580-68-5, C18H32O16

Refer to Structure Key on pages 2-4

[Link]

G 8281

Glycan and
Carbohydrate Standards

Blood Group Antigens

Glycan and Carbohydrate Standards

[Link]

Glycan and
Carbohydrate Standards

Blood Group Antigens

A 7911

A-Trisaccharide

Syn: Blood group A trisaccharide, a-L-Fuc-(12)-[a-D-GalNAc-(13)]-D-Gal

49777-13-1, C20H35NO15

B 1422

B-Trisaccharide

Syn: Blood group B trisaccharide, a-L-Fuc-(12)(a-D-Gal-[13])-D-Gal

49777-14-2, C18H32O15

F 7297

H-Trisaccharide

Syn: Blood Group H trisaccharide, a-Fuc-(12)-b-Gal-(14)-GlcNAc,

2-Fucosyl-N-acetyllactosamine
C2H35NO16

A 8956

A-Tetrasaccharide

Syn: a-D-GalNAc-(13)[a-L-Fuc-(12)]-b-D-Gal-(14)-D-Glc
59957-92-5, C26H45NO20

A 1955

A-Pentasaccharide

Syn: a-D-GalNAc-(13)-[a-L-Fuc-(12)]-b-D-Gal-(14)-[a-L-Fuc-(13)]-D-Glc
50624-46-9, C32H55NO24

Refer to Structure Key on pages 2-4

Glycan and Carbohydrate Standards

B 3791

B-Pentasaccharide

Syn: a-Fuc-(12)[a-Gal-(13)]-b-Gal-(14)[a-Fuc-(13)]-Glc
72468-43-0, C30H52O24

A 7560

iso-A-Pentasaccharide

Syn: a-GalNAc-(13)[a-Fuc-(12)]-b-Gal-(13)-[a-Fuc-(14)]-Glc,
A-Leb-Pentasaccharide
128464-25-5, C32H55NO24

B 0799

iso-B-Pentasaccharide

Syn: a-Fuc-(12)[a-Gal-(13)]-b-Gal-(13)[a-Fuc-(14)]-Glc,
b-Leb-Pentasaccharide
128464-26-6, C30H52O24

Glycan and
Carbohydrate Standards

Blood Group Antigens

L 7276

Lewis-a trisaccharide

Syn: a-L-Fuc-(14)-[b-D-Gal-(13)]-D-GlcNAc, Lea trisaccharide

56570-03-7, C20H35NO15

L 5152

Lewis-X trisaccharide

Syn: a-L-Fuc-(13)-[b-D-Gal-(14)]-D-GlcNAc, Lex trisaccharide

71208-06-5, C20H35NO15

Refer to Structure Key on pages 2-4

[Link]

Lewis and Cell Adhesion Glycans

Glycan and Carbohydrate Standards

[Link]

Glycan and
Carbohydrate Standards

Lewis and Cell Adhesion Glycans

L 7659

Lewis-b tetrasaccharide

Syn: a-Fuc(12)-b-Gal-(13)-[a-Fuc-(14)]-GlcNAc, Leb tetrasaccharide

C38H67NO29

L 7784

Lewis-Y tetrasaccharide

Syn: a-Fuc-(12)-b-Gal-(14)-[a-Fuc-(13)]-GlcNAc, LeY tetrasaccharide

C38H67NO29

L 7401

Lewis-Y hexasaccharide

Syn: a-Fuc-(12)-b-Gal-(14)(a-Fuc-[13])-b-GlcNAc-(13)-b-Gal-(14)-Glc, Ley-lactose,

Lacto-N-neo-difucohexaose I, LeY hexasaccharide
62469-99-2, C38H65NO29

S 2279

3-Sialyl-Lewis-a tetrasaccharide

Syn: a-NeuNAc-(23)-b-D-Gal-(13)-[a-L-Fuc-(14)]-D-GlcNAc, 3-SLea

92448-22-1, C31H52N2O32

S 1782

3-Sialyl-Lewis-X tetrasaccharide

Syn: a-NeuNAc-(23)-b-D-Gal-(14)[a-L-Fuc-(13)]-D-GlcNAc, 3-SLeX

98603-84-0, C31H52N2O23

Refer to Structure Key on pages 2-4

Glycan and Carbohydrate Standards

Gal a(1-3) Gal Antigens
G 7522

3a-Galactobiose

Syn: a-D-Gal-(13)-D-Gal, 3-O-a-D-Galactopyranosyl-D-galactose

13168-24-6, C12H22O11

G 9787

3a,4b-Galactotriose

Syn: a-D-Gal-(13)-b-D-Gal-(14)-D-Gal, 4-O-(3-O-a-D-Galactopyranosylb-D-galactopyranosyl)-D-galactopyranose

56038-36-9, C18H32O16

G 9912

3a,4b,3a-Galactotetraose

Syn: a-D-Gal-(13)-b-D-Gal-(14)-b-D-Gal-(13)-D-Gal, 3-O-(4-O-[3-O-a-D-Galactopyranosylb-D-galactopyranosyl]-a-D-galactopyranosyl)-D-galactopyranose

56038-38-1, C24H42O21

M 5107

IgM, Lambda
from murine myeloma

Clone MOPC 104E, Purified immunoglobulin, Buffered aqueous solution

M 2521

IgM, Lambda
from murine myeloma

Clone MOPC 104E, Ascites fluid, Lyophilized powder

G 7528

D-Glucose

Syn: Grape sugar, Dextrose, Corn sugar

50-99-7, SigmaUltra 99.5% (GC), C6H12O6

G 6404

D-Galactose

59-23-4, SigmaUltra minimum 99%, C6H12O6

M 8296

D-Mannose

Syn: D-Mannopyranose
3458-28-4, minimum 99% SigmaUltra, C6H12O6

F 2252

L-Fucose

Syn: 6-Deoxy-L-galactose
2438-80-4, minimum 99%, C6H12O5

X 3877

D-Xylose

58-86-6, SigmaUltra 99%, C5H10O5

A 8625

N-Acetyl-D-glucosamine

Syn: 2-Acetamido-2-deoxy-D-glucose, D-GlcNAc

7512-17-6, C8H15NO6

A 2795

N-Acetyl-D-galactosamine

Syn: D-GalNAc, N-Acetylchondrosamine, 2-Acetamido-2-deoxy-D-galactose

1811-31-0, C8H15NO6

A 0812

N-Acetylneuraminic acid

Syn: NeuNAc, Sialic acid, Lactaminic acid, NANA, 5-Acetamido-3,5-dideoxy-D-glyceroD-galactononulosonic acid, NAN
131-48-6, Type IV-S, C11H19NO9

G 2755

N-Glycolylneuraminic acid

Syn: NeuNGl
1113-83-3, C11H19NO10

G 8645

D-Glucuronic acid sodium salt

Syn: Sodium D-glucuronate

14984-34-0, C6H9NaO7 H2O

Glycan and
Carbohydrate Standards

Gal a(1-3) Gal Antigens

Monosaccharides

[Link]

Refer to Structure Key on pages 2-4

Glycobiology Product Directory

Additional Information Sources
Visit the Sigma-Aldrich Glycoprotein Analysis home page at [Link]/glyco
This new and growing web page contains:
Hundreds of enzymes, glycoproteins,
substrates, and reagents for glycoprotein
analysis
Links to additional enzymes, inhibitors,
antibodies, and lectins as well as related
products available through the
Enzyme Explorer
Product information, MSDS, and
Certificates of Analysis
For direct link to the most up-to-date
new product information visit

[Link]/glycogo

Glycobiology
Product Directory

Sigma-Aldrich Enzyme Explorer

[Link]/enzymeexplorer
Tap into the most comprehensive line of enzymes, substrates, and inhibitors in the industry. The Sigma Enzyme
Explorer is our online resource center that will simplify
finding the products and information you need.
A web-based search tool that allows you to find:
1,600 enzymes by name, application, EC number, or
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Over 1,000 links in the indices to inhibitors and substrates
organized by their corresponding enzymes
Enzyme assay procedures
Product Information Sheets, Certificates of Analysis,
and MSDS

Choose an index and find a product

The Enzyme Explorer offers the flexibility of four different
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Search option to find any product by name.
The Enzyme Application Index contains 35 different
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extracellular matrices, NOS, signal transduction, cell
dissociation and lysis, proteases for fusion proteins
and sequencing, PCR, restriction enzymes, and so on.
The Enzyme Class Index helps you find enzymes by their
EC number or by the type of reaction they catalyze.
The Enzyme Inhibitor Index contains over 600 inhibitor
product links organized by their corresponding enzymes.
The Enzyme Substrate Index contains approximately
400 substrate product links.

[Link]

Click on More Technical Data

Enzyme assay procedures containing all the information you need to perform the assay in your lab.
Our Product Information Sheets contain up-to-date
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For advice and information on our products, contact Sigma Technical Services by email at

techserv@[Link]

Enzymes
Complete List of Glycolytic Enzymes
A 9763

a-N-Acetylgalactosaminidase

From chicken liver, lyophilized powder, 5-20 units/mg protein

A 6680

N-Acetylneuraminic acid Aldolase

Expressed in E. coli

Amyloglucosidase

Cellulase

Chitinase

Chondroitinase
C 2905

Chondroitinase ABC

From Proteus vulgaris, lyophilized powder, 0.3-3 units/mg

C 3667

Chondroitinase ABC

From Proteus vulgaris, lyophilized powder, 50-250 units/mg

Driselase

F 6151

a-L-Fucosidase

From human placenta, ammonium sulfate suspension, minimum 2 units/mg

F 5884

a-L-Fucosidase

From bovine kidney, ammonium sulfate suspension, minimum 2 units/mg

Fucosidase

[Link]

Galactosidase
G 7163

G 0914

b1,6-Galactosidase

Recombinant, expressed in E. coli, buffered aqueous solution, positionally specific

G 3153

b-Galactosidase

Recombinant, expressed in E. coli, overproducing strain, lyophilized powder,

minimum 500 units/mg

G 4259

b-Glucuronidase

From Helix aspersa (garden snail), 250,000-500,000 units/g

Glycobiology
Product Directory

Glucuronidase

[Link]

Glucosidase

Enzymes
Complete List of Glycolytic Enzymes
G 9028

d4,5-Glycuronidase

From Flavobacterium heparinum, aqueous solution, 20 units/mg

G 1163

O-Glycosidase

From Streptococcus pneumoniae, recombinant, expressed in E. coli,

buffered aqueous solution

H 2125

Hemicellulase

From Aspergillus niger, 0.01-0.1 unit/mg solid

H 2519

Heparinase I

From Flavobacterium heparinum, lyophilized powder,

stabilized with approx. 25% bovine serum albumin, 200-600 units/mg

H 6512

Heparinase II

From Flavobacterium heparinum, lyophilized powder, 100-300 units/mg

H 8891

Heparinase III

From Flavobacterium heparinum, lyophilized powder, 200-600 units/mg solid

Heparinase

Hesperidinase
H 8137

Hesperidinase

From Aspergillus niger, lyophilized powder, minimum 1 unit/g

H 8510

Hesperidinase

From Penicillium sp., lyophilized powder, minimum 10 units/g

Glycobiology
Product Directory

From human neutrophils, minimum 95% (SDS-PAGE), lyophilized powder,

minimum 100,000 units/mg

L 1129

Lysozyme Agarose

From chicken egg white, lyophilized powder, 5,000-10,000 units/g

M 1266

Lysozyme Agarose

From chicken egg white, lyophilized powder, 5,000-10,000 units/g

L 2879

Lysozyme chloride form

From chicken egg white, grade VI, not dialyzed or lyophilized, approx. 60,000 units/mg

[Link]

Lysozyme

Mannosidase
M 7257

a-Mannosidase

M 2658

a-Mannosidase

Buffered aqueous solution, expressed in E. coli, Recombinant

M 9400

b1,4-Mannosidase, b-Mannosidase

From Helix pomatia, From snail acetone powder, ammonium sulfate suspension, 5-30 units/ml

From Canavalia ensiformis (jack bean), ammonium sulfate suspension, approx. 20 units/mg

Enzymes
Complete List of Glycolytic Enzymes
N 1385

Naringinase

From Penicillium decumbens, 300 units/g

N 7271

a2,3-Neuraminidase

From Streptococcus pneumoniae, buffered aqueous solution

N 5521

a2-(3,6)-Neuraminidase

From Clostridium perfringens (C. welchii), recombinant, expressed in E. coli,

buffered aqueous solution

N 8271

a2-(3,6,8,9)-Neuraminidase

From Arthrobacter ureafaciens, positionally specific, recombinant, expressed in E. coli,

buffered aqueous solution

N 3786

a2-(3,6,8,9)-Neuraminidase

Proteomics grade

N 4883

Neuraminidase Agarose

From Clostridium perfringens (C. welchii), type X-A, ammonium sulfate suspension,
20-30 units/g agarose

N 5254

Neuraminidase Agarose

From Clostridium perfringens (C. welchii), type VI-A, ammonium sulfate suspension

N 2133

Neuraminidase

From Clostridium perfringens (C. welchii), type X, lyophilized powder,

minimum 50 units/mg, 150-400 units/mg

N 2876

Neuraminidase

From Clostridium perfringens, 0.5-6 units/mg solid, From Vibrio cholerae, type III,
1-3 units/mg protein

N 5631

Neuraminidase

From Clostridium perfringens (C. welchii), type VIII, lyophilized powder, 10-20 units/mg

N 3001

Neuraminidase

From Clostridium perfringens (C. welchii), type VI, lyophilized powder,

2-5 units/mg protein (mucin), 6-10 units/mg

N 7771

Neuraminidase

From Salmonella typhimurium, recombinant, expressed in E. coli, lyophilized powder,

minimum 400 units/mg

N 3642

Neuraminidase

From Arthrobacter ureafaciens, buffered aqueous solution, minimum 8 units/ml

N 7885

Neuraminidase

From Vibrio cholerae, type III, 1-3 units/mg protein

N 6514

Neuraminidase

From Vibrio cholerae, type II, saline solution, 8-24 units/mg

C 6105

Novozyme 188

Liquid, minimum 250 units/g

P 7052

Pectin Lyase acidic enzyme

From Aspergillus niger, buffered aqueous glycerol solution, 50-150 units/mg

P 2611

Pectinase

From Aspergillus aculeatus, minimum 26,000 units/ml, Pectinex Ultra SPL

P 2736

Pectinase

From Aspergillus niger, minimum 3,000 units/ml, Pectinex 3X L

P 2401

Pectinase

from Rhizopus sp., crude powder, 400-800 units/g solid

P 0764

Pectinesterase

From orange peel, ammonium sulfate suspension, 50-350 units/mg

P 3026

Pectolyase

From Aspergillus japonicus, lyophilized powder, minimum 0.3 units/mg

P 5431

Pectolyase

From Aspergillus japonicus, lyophilized powder, minimum 2 units/mg

P 8375

Peroxidase, Horseradish

Essentially salt-free

G 5166

PNGase F

from Chryseobacterium (Flavobacterium) meningosepticum, buffered aqueous solution

P 3304

Polygalacturonase

From Aspergillus japonicus, ammonium sulfate suspension, 300-1,500 units/mg

P 5420

Pullulanase

From Klebsiella pneumoniae, ammonium sulfate suspension, minimum 5 units/mg

P 1067

Pullulanase

From Klebsiella pneumoniae, lyophilized powder, 10-30 units/mg

S 9754

Sulfatase

From abalone entrails, type VIII, lyophilized powder, 20-40 units/mg

S 1629

Sulfatase

From Aerobacter aerogenes, type VI, buffered aqueous glycerol solution,

10-20 units/ml, 2-5 units/mg

S 9626

Sulfatase

From Helix pomatia, type H-1, minimum 10,000 units/g solid

S 8504

Sulfatase

From Patella vulgata (keyhole limpet), type IV, essentially salt-free, lyophilized powder,
10 units/mg

Glycobiology
Product Directory

Neuraminidase

Pectinase

Pectolyase

Pullulanase

[Link]

Sulfatase

Enzymes
Complete List of Glycolytic Enzymes
Sulfatase (cont)
S 8629

Sulfatase

From Patella vulgata (keyhole limpet), type V, Essentially salt-free, lyophilized powder,
minimum 5 units/mg

S 1062

2-O-Sulfatase

From Flavobacterium heparinum, buffered aqueous solution, 10-40 units/ml

S 9751

Sulfatase

From Helix pomatia, type H-2, crude solution, minimum 2,000 units/ml

T 4528

Thioglucosidase

From Sinapis alba (white mustard) seed, minimum 175 units/g

T 8778

Trehalase

From porcine kidney, buffered aqueous glycerol solution, minimum 0.4 units/mg

V 2010

Viscozyme L

From Aspergillus sp., enzyme mixture containing carbohydrases, including arabanase,

cellulase, B-glucanase, hemi-cellulase and xylanase

GPI Anchor Enzymes

C 2557

Ceramide Glycanase

Buffered aqueous solution, minimum 0.01 units/mg

E 2277

Endoglycoceramidase II
plus activator II

From Rhodococcus sp., endo-type cleavage of cell surface glycosphingolipids of living cells
without damaging other cell components

P 5542

Phospholipase C,
Phosphatidylinositol-specific

From Bacillus cereus, lyophilized powder, 3,000 units/mg

Used for the release of GPI anchored proteins from the membrane

P 8804

Phospholipase C,
Phosphatidylinositol-specific

From Bacillus cereus, buffered aqueous glycerol solution, 3,000 units/mg

Used for the release of GPI anchored proteins from the membrane

P 5147

Pronase E

From Streptomyces griseus (protease), powder, type XIV, approx. 4 units/mg

A mixture of at least three proteolytic activities including an extracellular serine protease

[Link]

Glycosyltransferases
74188

a-1,3-Galactosyl-Transferase Kit

a-1,3-Galactosylation kit, contains glycosyltransferase, nucleotide sugar (donor), buffer

and supplementary reagents for performing five glycosylations

77038

a-1,3-Galactosyl-Transferase

Recombinant, expressed in E. coli, solution, ~0.5 units/ml

Enzyme employed for the chemoenzymatic synthesis of trisaccharides from disaccharides in
quantitative yield; synthesis of xenoactivea-galactosyl epitopes

59505

b-1,4-Galactosyl Transferase Kit

b-1,4-Galactosylation Kit

90261

b-1,4-Galactosyl Transferase I

Recombinant human, expressed in Saccharomyces cerevisiae

Enzyme catalyst for the stereo-selective synthesis of galactosyl carbohydrates

48279

Galactosyltransferase

From bovine milk, lyophilized powder, ~1 units/mg

48281

Galactosyltransferase

From bovine milk, lyophilized powder, ~8 units/g

Catalyst for the chemoenzymatic oligosaccharide synthesis, e.g. transfer of galactose

Inhibitors
Inhibitors of Carbohydrate Metabolism
P 7340

()-threo-1-Phenyl-2-decanoyl-amino-3-morpholino-1-propanol hydrochloride

Syn: PDMP

H 9632

(2R,5R)-Bis(hydroxymethyl)-(3R,4R)-dihydroxypyrrolidine

Syn: DMDP

D 8390

1,4-Dideoxy-1,4-imino-D-mannitol hydrochloride

Minimum 90% by HPLC

D 9160

1-Deoxymannojirimycin hydrochloride

Syn: 1,5-Dideoxy-1.5-imino-D-mannitol HCl

D 9305

1-Deoxynojirimycin hydrochloride

Syn: 1,5-Dideoxy-1,5-imino-D-sorbitol HCl

A 5416

2-Acetamido-1,2,5-trideoxy-1,5-imino-D-glucitol

Minimum 90% by HPAE

D 8875

2-Deoxy-D-glucose 6-phosphate sodium salt

Minimum 95% by TLC

Deoxyglucose
D 8375

2-Deoxy-D-glucose

Grade II, crystalline

D 6134

2-Deoxy-D-glucose

Grade III, crystalline

D 3179

2-Deoxy-D-glucose

SigmaUltra

B 1147

6-Bromo-4-cyclohexene-1,2,3-triol

Mixed isomers

B 6676

7(S)-Brefeldin A-7-3H

Ethanol solution

B 4894

Benzyl 2-acetamido-2-deoxy-a-D-galactopyranoside

Syn: a-D-GalNAc-1OCH2Ph

B 6542

Brefeldin A

From Penicillium brefeldianum,

for molecular biology, minimum 98% (TLC)

B 7651

Brefeldin A

From Penicillium brefeldianum,

minimum 99% (TLC)

C 3784

Castanospermine

From Castanospermum australe seeds

27689

Concanamycin A

From Streptomyces sp., BioChemika

C 9705

Concanamycin A

From Streptomyces sp., syn: Folimycin

C 5424

Conduritol B epoxide

Syn: 1,2-Anhydro-myo-inositol

D 9641

Deoxygalactonojirimycin hydrochloride

Syn: 1,5-Dideoxy-1,5-imino-D-galactitol

P 4194

DL-threo-1-Phenyl-2-palmitoyl-amino-3-morpholino-1-propanol

Syn: ()-PPMP, minimum 98% by TLC

69895

Monensin decyl ester, Selectophore

100 mg/ml in tetrahydrofurane

M 5273

Monensin sodium salt

Syn: Monensin A

D 9050

N-Acetyl-2,3-dehydro-2-deoxyneuraminic acid

Approx. 95% by TLC

N 8028

Nikkomycin Z

From Streptomyces tendae

B 8299

N-Butyldeoxynojirimycin

Approx. 95% by TLC

M 1777

N-Methyl-1-deoxynojirimycin

Minimum 98% by HPLC

S 6640

Swainsonine

From Rhizoctonia leguminicola

S 9263

Swainsonine

Synthetic

T 0527

Tunicamycin A1 homolog

Approx. 95%

T 1152

Tunicamycin C2 homolog

Approx. 95% (HPLC)

T 7765

Tunicamycin

From Streptomyces sp.

Brefeldin

Glycobiology
Product Directory

Con A

Monensin

Nojirimycin

Swainsonine

[Link]

Tunicamycin

Substrates
Glycolytic Enzyme Substrates
Acetylneuraminic
M 8639

2-(4-Methylumbelliferyl)-a-D-N-acetylneuraminic acid

Minimum 95% (HPLC), sodium salt hydrate

N 1266

2-O-(o-Nitrophenyl)-a-D-N-acetylneuraminic acid

Approx. 95%

N 1516

2-O-(p-Nitrophenyl)-a-D-N-acetylneuraminic acid

Approx. 95%

B 4666

5-Bromo-4-chloro-3-indolyl a-D-N-acetylneuraminic acid

Minimum 90%, sodium salt

Arabinofuranoside
N 3641

4-Nitrophenyl a-L-arabinofuranoside

N 3512

4-Nitrophenyl a-L-arabinopyranoside

Minimum 99% (TLC)

N 0520

4-Nitrophenyl b-L-arabinopyranoside

Minimum 98% (TLC)

Cellobioside
N 4764

2-Nitrophenyl b-D-cellobioside

Minimum 95% (HPLC)

M 6018

4-Methylumbelliferyl b-D-cellobioside

Minimum 98% (TLC)

N 5759

4-Nitrophenyl b-D-cellobioside

Minimum 98% (HPLC)

B 6795

5-Bromo-4-chloro-3-indolyl b-D-cellobioside

Syn: X-Cellobiose

N 0145

4-Nitrophenyl b-D-cellopentaoside

Minimum 90%, powder

N 1262

4-Nitrophenyl b-D-cellotetraoside

Minimum 75%

N 4889

4-Nitrophenyl b-D-cellotrioside

Approx. 95%

M 9763

4-Methylumbelliferyl b-D-N,N-diacetylchitobioside

Minimum 98% (TLC), hydrate

N 6133

4-Nitrophenyl N,N-diacetyl-b-D-chitobioside

Minimum 99% (TLC)

N 8638

4-Nitrophenyl b-D-N,N,N-triacetylchitotriose

Minimum 98% (TLC)

M 5639

4-Methylumbelliferyl b-D-N,N,N-triacetylchitotrioside

Hydrate, catalyzed by chicken lysozyme, lipase

Chitobioside

Glycobiology
Product Directory

Fucopyranoside
N 3628

4-Nitrophenyl a-L-fucopyranoside

Minimum 98% (TLC)

N 3378

4-Nitrophenyl b-D-fucopyranoside

Minimum 98% (TLC)

N 2505

4-Nitrophenyl b-L-fucopyranoside

Minimum 99% (TLC)

B 9511

5-Bromo-4-chloro-3-indolyl b-D-fucopyranoside

Minimum 98%

B 2280

5-Bromo-4-chloro-3-indolyl b-L-fucopyranoside

Minimum 98%

N 3253

2-Nitrophenyl b-D-fucopyranoside

Minimum 99% (TLC)

M 5510

4-Methylumbelliferyl b-D-fucoside

Minimum 98%

M 4508

4-Methylumbelliferyl b-L-fucoside

Minimum 98%

Fucoside

[Link]

N 2257

4-Nitrophenyl 1-thio-b-D-galactopyranoside

Minimum 95%

N 2766

4-Nitrophenyl 2-acetamido-2-deoxy-3-O-(2-acetamido-2-deoxyb-D-glucopyranosyl)-a-D-galactopyranoside

Minimum 98%

N 3016

4-Nitrophenyl 2-acetamido-2-deoxy-3-O-(b-D-galactopyranosyl)a-D-galactopyranoside

Minimum 98%

N 7643

4-Nitrophenyl 6-O-b-D-galactopyranosyl-b-D-galactopyranoside

Minimum 95%

N 0877

4-Nitrophenyl a-D-galactopyranoside

Minimum 99% (TLC)

Substrates
Glycolytic Enzyme Substrates
Galactopyranoside (cont)
N 1252

4-Nitrophenyl b-D-galactopyranoside

Minimum 98%

T 3661

4-Trifluoromethylumbelliferyl b-D-galactopyranoside

Minimum 98% (TLC)

B 2904

5-Bromo-3-indolyl b-D-galactopyranoside

Molecular biology grade

B 4387

5-Bromo-3-indolyl b-D-galactopyranoside

Minimum 98%

B 6024

5-Bromo-4-chloro-3-indolyl b-D-galactopyranoside

5 mg tablet

B 9146

5-Bromo-4-chloro-3-indolyl b-D-galactopyranoside

Minimum 98%

F 6542

Fluorescein mono-b-D-galactopyranoside

Approx. 90%

I 7513

Indoxyl b-D-galactopyranoside

Minimum 98%

N 2885

Naphthol AS-BI b-D-galactopyranoside

Minimum 99% (TLC)

N 0257

2-Nitrophenyl N-acetyl-a-D-galactosaminide

Minimum 99% (TLC)

N 3273

2-Nitrophenyl-N-acetyl-b-D-galactosaminide

Minimum 99% (TLC)

M 9659

4-Methylumbelliferyl N-acetyl-b-D-galactosaminide

Minimum 98% (TLC)

N 4264

4-Nitrophenyl N-acetyl-a-D-galactosaminide

Minimum 98%

N 9003

4-Nitrophenyl N-acetyl-b-D-galactosaminide

Minimum 98%

B 3166

5-Bromo-4-chloro-3-indolyl N-acetyl-b-D-galactosaminide

Approx. 95%

Glycobiology
Product Directory

Galactosaminide

Galacturonide
N 8755

4-Nitrophenyl b-D-galacturonide

Minimum 98% (TLC)

Glucopyranoside
N 2007

2-Naphthyl b-D-glucopyranoside

N 8016

2-Nitrophenyl b-D-glucopyranoside

Minimum 99% (TLC)

M 9766

4-Methylumbelliferyl a-D-glucopyranoside

Minimum 99% (TLC)

Minimum 98% (TLC)

M 3633

4-Methylumbelliferyl b-D-glucopyranoside

Minimum 99% (TLC)

M 0662

4-Methylumbelliferyl-7-(6-sulfo-2-acetamido-2-deoxyb-D-glucopyranoside)

Approx. 98% (TLC), sodium salt

N 2381

4-Nitrophenyl 1-thio-b-D-glucopyranoside

Approx. 95%

N 5513

4-Nitrophenyl 2-acetamido-2-deoxy-3-O-b-D-galactopyranosylb-D-glucopyranoside

Minimum 98% (TLC)

N 1377

4-Nitrophenyl a-D-glucopyranoside

Minimum 99%

N 7006

4-Nitrophenyl b-D-glucopyranoside

Minimum 98% (TLC)

B 4527

5-Bromo-4-chloro-3-indolyl b-D-glucopyranoside

Approx. 98%

36937

6,8-Difluoro-4-methylumbelliferyl-b-D-glucopyranoside

98.0% (TLC), BioChemika

F 4521

Fluorescein di-(b-D-glucopyranoside)

Minimum 90%

M 9881

4-Methylumbelliferyl N-acetyl-a-D-glucosaminide

Minimum 98% (TLC)

M 2133

4-Methylumbelliferyl N-acetyl-b-D-glucosaminide

Minimum 98% (TLC), dihydrate

N 8759

4-Nitrophenyl N-acetyl-a-D-glucosaminide

Minimum 98%

N 9376

4-Nitrophenyl N-acetyl-b-D-glucosaminide

98-100%

B 3041

5-Bromo-4-chloro-3-indolyl N-acetyl-b-D-glucosaminide

Minimum 98%

N 4006

Naphthol AS-BI N-acetyl-b-D-glucosaminide

Minimum 95% (TLC)

I 6893

Indoxyl b-D-glucoside

SigmaUltra, minimum 97%

I 3750

Indoxyl b-D-glucoside

Minimum 97%

Glucoside

[Link]

Glucosaminide

Substrates
Glycolytic Enzyme Substrates
Glucuronide
N 9013

1-Naphthyl b-D-glucuronide

N 2645

2-Nitrophenyl b-D-glucuronide

Minimum 98% (TLC), potassium salt

M 9130

4-Methylumbelliferyl b-D-glucuronide

Minimum 98% (TLC), hydrate

M 5664

4-Methylumbelliferyl b-D-glucuronide

SigmaUltra, hydrate

N 1627

4-Nitrophenyl b-D-glucuronide

Minimum 98% (TLC)

T 6410

4-Trifluoromethylumbelliferyl glucuronide

Minimum 98% (TLC), potassium salt

B 8049

5-Bromo-4-chloro-3-indolyl b-D-glucuronide

10 mg tablet, cyclohexylammonium salt

B 0522

5-Bromo-4-chloro-3-indolyl b-D-glucuronide

Minimum 98%, cyclohexylammonium salt

B 3783

5-Bromo-4-chloro-3-indolyl b-D-glucuronide

Minimum 98%, cyclohexylammonium salt

B 6650

5-Bromo-4-chloro-3-indolyl b-D-glucuronide

Minimum 98% (TLC), cyclohexylammonium salt

B 8174

5-Bromo-4-chloro-3-indolyl b-D-glucuronide

10 mg tablet, sodium salt

B 5285

5-Bromo-4-chloro-3-indolyl b-D-glucuronide

Minimum 98%, sodium salt

B 4782

5-Bromo-4-chloro-3-indolyl b-D-glucuronide

Minimum 98% (TLC), sodium salt

B 4532

5-Bromo-6-chloro-3-indolyl b-D-glucuronide

Minimum 98%, cyclohexylammonium salt

B 4657

5-Bromo-6-chloro-3-indolyl b-D-glucuronide

Minimum 98%, cyclohexylammonium salt

36939

6,8-Difluoro-4-methylumbelliferyl-b-D-glucuronide

BioChemika, for fluorescence, 98.0% (TLC), lithium salt

24907

6-Chloro-3-indolyl-b-D-glucuronide

BioChemika, 97.0% (HPLC), cyclohexylamine salt

I 7638

Indoxyl b-D-glucuronide

Cyclohexylammonium salt

N 1875

Naphthol AS-BI b-D-glucuronide

Approx. 95%, sodium salt

P 0376

Phenolphthalein b-D-glucuronide

Sodium salt

P 0501

Phenolphthalein b-D-glucuronide

Free acid

Approx. 99%, sodium salt

Glycobiology
Product Directory

Approx. 98% (TLC)

M 0905

4-Methylumbelliferyl b-D-mannopyranoside

Minimum 98% (TLC)

N 2127

4-Nitrophenyl a-D-mannopyranoside

Minimum 98% (TLC)

N 1268

4-Nitrophenyl b-D-mannopyranoside

Approx. 98%

B 4526

5-Bromo-4-chloro-3-indolyl a-D-mannopyranoside

Approx. 95%

G 2000

2-(b-D-Galactosidoxy)naphthol AS-LC

Minimum 95% (TLC)

A 8750

N-Acetyl-b-D-glucosamine naphthol AS-LC

Minimum 95% (TLC)

D-()-Salicin

Minimum 99%, substrate for b-Galactosidase

Naphthol

Salicin
S 0625

Rhamnopyranoside
[Link]

2-Amido-GalN-BSA

A 2420

a-Gal-FITC-BSA

A 6544

a-Gal-TRITC-BSA

A 8165

b-Gal-FITC-BSA

A 1159

Aminophenyl-b-GalNAc-BSA

Glucose
A 6158

GlcNAc-BSA

A 5543

a-Glc-FITC-BSA

A 1034

Aminophenyl-b-GlcNAc-BSA

A 7172

b-Glc-FITC-BSA

A 6052

GlcNAc-resorufin-BSA

Fructose
A 8426

Fructosamine-BSA (glycated)

A 8301

Fructosamine-HSA (glycated)

A 4042

1-Amido-Fuc-biotin-BSA

A 6033

1-Amido-Fuc-BSA

A 5793

a-L-Fuc-FITC-BSA

A 5918

a-L-Fuc-TRITC-BSA

Glycobiology
Product Directory

Fucose

Lactose
A 8210

Lactose-BSA

A 7799

b-Lactose-biotin-BSA

A 8040

b-Lactose-FITC-BSA

A 7665

b-Lactose-TRITC-BSA

A 5783

Lactitol-1-N-BSA

Maltose
A 5283

Maltitol-1-N-BSA

A 8460

Maltose-BSA

Mannose
A 7924

a-Man-biotin-BSA

A 8303

a-Man-BSA

A 7790

a-Man-FITC-BSA

A 7915

a-Man-TRITC-BSA

A 4664

Aminophenyl-a-Man-BSA

A 3955

b-Xyl-FITC-BSA

[Link]

Xylose

Lectins
Lectins are proteins or glycoproteins from non-immune origins that agglutinate cells and/or precipitate complex carbohydrates. The agglutination activity of these highly specific carbohydrate-binding molecules is usually inhibited by a
simple monosaccharide, but for some lectins di-, tri-, and even polysaccharides are required.
Sigma offers a wide range of lectins suitable for the following applications:
Carbohydrate studies
Lymphocyte subpopulation studies
Blood group typing
Fractionation of cells and other particles
Mitogenic stimulation
Histochemical studies

[Link]

Glycobiology
Product Directory

Lectins are isolated from a wide variety of natural sources. Sigma offers a range of lectins suitable for all of the
applications noted above.
Common Name

Taxonomic Name

Common Name

Taxonomic Name

Abrin

Abrus precatorius

Labumum, Scotch

Labumum polyphemus

Asparagus pea

Tetragonolobus purpureas

Lentil

Lens culinaris

Avocado

Perseau americana

Lotus

Tetragonolobus purpureas

Bitter pear melon

Momordica charantia

Mistletoe, European

Viscum album

Broad bean

Vicia faba

Mung bean

Vigna radiata

Camels foot tree

Bauhinia purpurea

Mushroom

Agaricus bisporus

Castor bean

Ricinus communis

Osage orange

Maclura pomifera

Chick pea

Cicer arietinum

Pagoda tree

Sophora japonica

Cobra, Mozambique

Naja mocambiqu mocambique

Pea, garden

Pisum sativum

Cobra, Thailan

Naja naja kaouthis

Peanut

Arachis hypogaea

Con A

Canavalia ensiformis

Pokeweed

Phytolacca americana

Concanavalin A

Canavalia ensiformis

Potato

Solanum tuberosum

Coral tree, Israel

Erythrina corallodendron

Red kidney bean

Phaseolus vulgaris

Daffodil

Narcissus pseudonarcissus

Red marine algae

Ptilota plumosa

Eel

Anguilla anguilla

Roman snail

Helix pomatia

Elder

Sambucus nigra

Scarlet runner bean

Phaseolus coccineus

Fava bean

Vicia faba

Siberian pea tree

Caragana arborescens

Furze

Ulex europaeus

Snail, garden

Helix aspersa

Gorse

Ulex europaeus

Snowdrop

Galanthus nivalis

Green marine algae

Codium fragile

Soybean

Glycine max

Hairy vetch

Vicia villosa

Spindle tree

Euonymus europaeus

Horse gram

Dolichos biflorus

Sweet pea

Lathyrus odoratus

Horseshoe crab

Limulus polyphemus

Thorn Apple

Datura stramonium

Jacalin

Artocarpus intefrifolia

Tomato

Lycopersicon esculentum

Jack bean

Canavalia ensiformis

Wheat germ

Triticum vulgaris

Japanese wisteris

Wisteria floribanda

Winged bean

Psophocarpus tetragonolobus

Jequity bean

Abrus precatorius

Winged pea

Tetragonolobus purpureas

Jimson weed

Datura stramonium

Many lectins are available as conjugates. Conjugation does not alter the specificity of the lectin. The degree of conjugation
is expressed as 1) a mole of dye per mole of lectin basis 2) a unit of enzyme activity per mg of protein or 3) a mg of
protein per ml of packed gel basis. For colloidal gold conjugates, the activity of the lectin is determined by a dot blot
assay using an appropriate glycoprotein. Agglutination activity is expressed in microgram per ml and is determined
from serial dilution studies of a 1 mg per ml solution. This activity is the lowest concentration of lectin to agglutinate
a 2% suspension of appropriate erythrocytes after 1 hour incubation at 25 C.

Lectins
Lectins
Product
Number

Source

Mol. Wt. (kDa)

Subunits

Specificity
Blood Group

Specificity Sugar

L 5640

Agaricus bisporus

58.5

b-Gal(13)GalNAc

L 0881

Arachis hypogaea

120

b-Gal(13)GalNAc

a-GalOMe

114

A,B

a-Gal, a-GalNAc

114

a-Gal

L 7759

Peroxidase

L 6135

Biotin

L 7381

FITC

L 3766
L 3515

Mitogenic
Activity

TRITC
Artocarpus integrifolia

L 4650

Peroxidase

L 5147

Agarose

(+)

BS-I

L 3759

Biotin

L 9381

FITC

L 5264

TRITC

L 0890

FITC

L 3019

BS-I-B4

L 5391

Peroxidase

L 2140

Biotin

L 2895

FITC

L 9637

Caragana arborescens

60;120(c)

2;4

GalNAc

L 3141

Cicer arietinum

fetuin

L 2638

Codium fragile

GalNAc

C 7275

Concanavalin A

102

a-Man, a-Glc

(+)

L 6397

Peroxidase

C 2272

Biotin

C 7642

FITC

C 6904

Agarose

C 9017

Sepharose

L 5021

Gold, 10 nm

L 3642

Gold, 20 nm

C 7898

Ferritin
51

a-Man, a-Glc

(+) (d)

L 9385

Succinyl-Concanavalin A
FITC

L 2766

Datura stramonium

2(a and b)(a)

(GlcNAc)2

L 2785

Dolichos biflorus

140

a-GalNAc

L 1287

Peroxidase

L 6533

Biotin

L 9142

FITC

L 9658

TRITC

L 9894

Agarose

L 5390

Erythrina cristagalli

56.8

2(a and b)(a)

b-Gal(14)GlcNAc

L 7400

Euonymus europaeus

166

4(a and b)(a)

B,H

a-Gal(13)Gal

L 8725

Galanthus nivalis

(h)

non-reduc. D-Man

L 8775

Agarose

(+)

[Link]

L 3885

Glycobiology
Product Directory

Bandeiraea simplicifolia
L 2380

Lectins
Lectins
Product
Number

Source

Mol. Wt. (kDa)

Subunits

Specificity
Blood Group

Specificity Sugar

Mitogenic
Activity

L 1395

Glycine max

110

GalNAc

(+) (b)

L 2650

Peroxidase

L 4511

TRITC
79

GalNAc

a-Man

NeuNAc

L 6635
L 8764

Biotin

L 3382

Helix pomatia

L 6387

Peroxidase

L 6512

Biotin

L 1034

FITC

L 1261
L 9267

TRITC
Lens culinaris

L 4143

Biotin

L 9262

FITC

L 0511

Limulus polyphemus

400

L 2886

Lycopersicon esculentum

L 0401
L 8025
L 4401
L 3138
L 4389

(+)

Agarose

L 2263

L 0651

Glycobiology
Product Directory

Helix aspersa

(GlcNAc)3

(+) (e)

sialic acid

(+)

Biotin
FITC
Maackia amurensis

130

2(a and b)

112

128

oligosaccharide

(+)

126

oligosaccharide

(+)

(GlcNAc)3

(+)

a-Man

(+)

Peroxidase
Phaseolus coccineus
TRITC
Phaseolus vulgaris

L 8629
L 6139

PHA-E
TRITC

L 2769

PHA-L

L 8754

PHA-P

L 2646

PHA-M

L 9379

Phytolacca americana

32(f)

L 5380

Pisum sativum

L 0770

4(a and b)(a)

GalNAc, Gal

120

b-Gal

FITC

L 9895

Pseudomonas aeruginosa (PA-I) 13-13.7

L 2138

Psophocarpus tetragonolobus

L 3139

Peroxidase

L 3014

Biotin

L 3264

Gal

FITC
Ricinus communis

[Link]

L 7886

Agglutinin, RCA120

L 2758

Peroxidase

L 9514

Ricin, A chain

L 4022

Ricin, A chain, deglycosylated

L 9639

Ricin, B chain

L 6890

Sambucus nigra

L 4266

Solanum tuberosum

Lectins
Lectins
Product
Number

Source

Mol. Wt. (kDa)

Subunits

Specificity
Blood Group

Specificity Sugar

L 9254

Tetragonolobus purpureas

120(A), 58(B),
117(C)

4;2;4

a-L-fuc

L 5759

Peroxidase

L 3134

Biotin

L 5644

FITC

L 9640

Triticum vulgaris

L 3892

Peroxidase

L 0390

Peroxidase (inactive)

L 5142

Biotin

L 4895

FITC

L 9884

Evans Blue

L 1894

Gold, 10 nm

L 1882

Agarose

(GlcNAc)2, NeuNAc

Mitogenic
Activity

(+)

L 8146

Peroxidase

L 8262

Biotin

L 9006

FITC

L 4889

TRITC

L 4011

Vicia villosa

L 9388

Agarose

L 7513

Isolectin B4

L 7888

Agarose

L 2662

a-L-Fuc

139

4(a)

A1+Tn

GalNAc

143

GalNAc

Viscum album

115 g

4(a and b)(a)

b-Gal

Wisteria floribunda

GalNAc

L 1516

Biotin

L 2016

Reduced

L 1766

Biotin

Notes:
a Subunits are of different molecular weights
b Mitogenic for neuraminidase-treated lymphocytes
c Concentration-dependent mol. wt. change
d Non-agglutinating and mitogenic

e Inhibits mitogenic activity of PHA

f Data given for PWM Pa2
h Agglutinates rabbit, but not human, erythrocytes

[Link]

UEA I

Glycobiology
Product Directory

Ulex europaeus
L 5505

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Glycoprotein
Analysis
Glycan
Classification
Glycoprotein
Purification
Deglycosylation
Strategies
Detection

Glycoprotein Analysis
Application Note
PNGase F Enzyme:
Efficient N-Deglycosylation
in Glycoprotein Analysis
By Elner Rathb

Overview
Key to Monosaccharide Symbols,
Abbreviations and Projections . . . . . . . . . . . . 02
Classification and Structur

2
s i g m a - a l d r i c h . c o m
Key to Monosaccharide Symbols, Abbreviations, and Projections
Symbols, Structure Projecti

3
s i g m a - a l d r i c h . c o m
Key to Monosaccharide Symbols, Abbreviations, and Projections
Symbols, Structure Projecti

4
s i g m a - a l d r i c h . c o m
Key to Monosaccharide Symbols, Abbreviations, and Projections
Symbols, Structure Projecti

5
s i g m a - a l d r i c h . c o m
Basic N-linked Structure
Classification and Structure of Glycan Components
N-Linked Glyca

6
s i g m a - a l d r i c h . c o m
High-Mannose Structure

7
s i g m a - a l d r i c h . c o m
Hybrid Structure

8
s i g m a - a l d r i c h . c o m
Complex Structure (tetraantennary)

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