ISOELECTRIC FOCUSING

 Electrophoretic method that separates

proteins according to the iso-electric
points
 Is ideal for seperation of amphoteric

substances
 Seperation is achieved by applying a

potential difference across a gel that

contain a pH gradient
 Isoelectric focusing requires solid support

such as agarose gel and polyacrylamide

gel
IEF
Separates proteins by their
isoelectric points (pI)
Each protein has own pI = pH
at which the protein has equal
amount of positive and
negative charges (the net
charge is zero)
IEF example

IEF 4-6.5 pH
gradient
Zavialov A.
IEF
Mixtures of ampholytes, small
amphoteric molecules with high
buffering capacity near their pI, are
used to generate the pH gradient.
Positivelyand negatively charged
proteins move to – and +,
respectively, until they reach pI.
PIof proteins can be theoretically
predicted. Therefore, IEF can also
be used for protein identification.
Principle
Separation of amphoteric substances
such as proteins

Separation of molecules according to

their different isoelectric points
Principle
High resolution
• pI difference 0.01 of a pH unit

Utilizes horizontal gels on

• Glass plates or
• Plastic sheets
Separation
Applying a potential difference
across a gel that contains a pH gradient
Ampholytes
The pH gradient is formed by
ampholytes

Complex mixtures of
• Synthetic polyaminopolycarboxylic
acids
Ampholytes: pH Gradient
Range
Wide band (e.g. pH 3-10) or
Narrow bands (e.g. pH 7-8), and
pI values should lie within this range

Commercially available
• Bio-Lyte and
• Pharmalyte
Thickness
Traditionally 1-2 mm thick IEF gels
• Relatively high cost of ampholytes
Thickness
Thin-layer IEF gels (0.15 mm thick)
Prepared using a layer of electrical
insulation tape
• As spacer between gel plates
Gel Percentage
Low percentage gels to avoid
• Any sieving effect within the gel
Gel Percentage
Polyacrylamide gels (4%)
Agarose is also used for high Mr
proteins
Gel Preparation
Ampholytes and riboflavin
• Mixed with the acrylamide solution

Mixture is poured over

Glass plate (typically 25 cm × 10 cm)
which
• Contains the spacer
Gel Polymerization
Second glass plate is placed on top of
the first
• To form the gel cassette
Gel polymerised by photo-
polymerisation by placing the gel in
front of a bright light
Gel Polymerization
Photodecomposition of the
riboflavin generates a free radical,
which
• Initiates polymerisation
Takes 2-3 h

Glass plates are prised apart

pH Gradient preparation
Electrode wicks: thick (3 mm) strips of
wetted filter paper
• Laid along the long length of each side
of the gel
pH Gradient preparation
• Anode is phosphoric acid H3PO4
• Cathode sodium hydroxide

Potential difference applied

pH Gradient preparation
Ampholytes form a pH gradient
between the anode and cathode
• The power is then turned off
Sample Application
By laying on gel small squares of filter
paper soaked in the sample
• A voltage is again applied for about
30 min to
• Allow the sample to electrophorese
off the paper and into the gel
Paper squares can be removed from
the gel
Sample Movement
Depending on which point on the pH
gradient the sample has been loaded
Positively Charged Proteins
Proteins that are initially at a pH region
below their isoelectric point will be
• Positively charged
• Will initially migrate towards the
cathode
Positively Charged Proteins
As they proceed, surrounding pH will
be steadily increasing
• Positive charge on the protein will
decrease correspondingly
Protein arrives at a point where the
pH is equal to its isoelectric point
Positively Charged Proteins
The protein will now be in the
zwitterion form with no net charge, so
further movement will cease.
Negatively Charged Proteins
Substances that are initially at pH
regions above their isoelectric points
will be negatively charged
• Will migrate towards the anode until
they reach their isoelectric points and
become stationary
Separation of Proteins
Movement does not depend upon place
of application

For rapid separations (2-3 h)

• Relatively high voltages (up to 2500
V) are used
Countering Heat Production
Gels are run on cooling plates (10 °C)
Stabilised power output
Indirect Staining
Followed because ampholytes will stain
too
• Giving a totally blue gel

Gel washed with fixing solution

• 10% (v/v) trichloroacetic acid
Staining: TCA
Precipitates proteins in gel
Allows smaller ampholytes to be
washed out

Gel is stained with Coomassie

Brilliant Blue and then destained
pI Detection
By running a mixture of proteins of
known pI on same gel

Available commercially
• Covering the pH range 3.5-10
pI Detection by Known
Proteins
Distance of each band from one
electrode is measured

Graph of distance for each protein

against its pI (effectively the pH at that
point) plotted
pI Detection by Known
Proteins
By this calibration line

pI of an unknown protein can be

determined by its position on gel
Applications:
Microheterogeneity
A protein may show a single band on
an SDS gel
May show three bands on an IEF gel
• May occur when a protein exists in
mono-, di- and tri-phosphorylated
forms
• Affects pI not Mr
Applications: Separating
Isoenzymes
Different forms of the same enzyme
often differing by only one or two
amino acid residues
Applications: Separating
Isoenzymes
Detected in the gel by
• Washing the unfixed and unstained
gel in an appropriate substrate OR
• Overlayering with agarose containing
the substrate

May be used for forensic purposes

Applications: Vertical column
IEF
Preparative
Water-cooled vertical glass column
Filled with a mixture of ampholytes
• Dissolved in sucrose solution

Density gradient to prevent diffusion

Applications: Vertical column
IEF
After separation
Sample components run out through a
valve in base of column

Can be carried out in beds of

granulated gel (Sephadex G-75)
 2-D Gel
Electrophoresis
Principle
Combines
• IEF (first dimension)
• SDS–PAGE (second dimension)

Gives two-dimensional (2-D) PAGE

Large format 2-D gels (20 cm × 20
cm)
Minigel system can be used
Procedure: First Dimension
(IEF)
Carried out in an acrylamide gel
• Cast on a plastic strip (18 cm ×
3mm)

Gel contains
• Ampholytes (for forming the pH
gradient)
• 8M urea and a non-ionic detergent
Procedure: First Dimension
(IEF)
Urea and detergent denature and
maintain solubility of proteins

Denatured proteins separate according

to their pI

After the run, IEF strip is incubated in a

sample buffer containing SDS
Procedure: Second Dimension
(SDS-PAGE)
Placed between the glass plates and on
top of
• Previously prepared 10% SDS–PAGE
gel
Procedure: Second Dimension
(SDS-PAGE)
Electrophoresis is commenced
SDS-bound proteins run into the gel
Separate according to size
Procedure: Time Factor
IEF gels are provided as dried strips
• Need rehydrating overnight

IEF run takes 6-8 h

Equilibration step with SDS sample
buffer takes about 15 min
SDS–PAGE step takes about 5 h
Efficacy
Can routinely resolve between 1000
and 3000 proteins from a cell or
tissue extract

Reported separation of between 5000

and 10000 proteins
Applications: Gene
Expression Change
Between two different biological
states
• Normal and diseased tissue

2-D Gel pattern of extract from a

diseased tissue (liver tumour)
compared with same of normal liver
tissue to observe pattern change
Applications: Gene
Expression Change
Presence (or absence) of proteins in
the liver tumour sample
By identifying this protein expression
pattern can be compared for two states

Molecular basis of diseases

Applications: Gene
Expression Change
Normal and diseased tissue can be
compared for
• Arthritis
• Kidney disease
• Heart valve disease

Significant proportion of change because

of high efficiency of separation
Applications: Analysis of
Treatment
Comparing normal with diseased tissue
we can:
• Analyse effects of drug treatment or
toxins on cells
• Observe changing protein component
of cell at different stages of tissue
development
Applications: Analysis of
Treatment
• Observe response to extracellular
stimuli such as hormones or cytokines
• Compare pathogenic and non-
pathogenic bacterial strains
Applications: Establishing
Biomarkers
Compare serum protein profiles from
healthy vs patients

Can then be developed as diagnostic

markers for diseases

Setting up ELISA to measure the specific

protein
• [Link]
procedure

[Link] principle
• At pH below its isoelctric point, proteins would be
a) Positively charged
b) Negatively charged
• In 2 dimensional gel electrophoresis, proteins
separate on the basis of
A) Isoelectric point
B) Mass
C) both