Protein Extraction and Quantification

A. Aims:
In this lab session, you will extract and quantify proteins from cells in culture.

B. Learning Objectives:
1. Understanding the basics of protein extraction, quantification and purification
methods
2. Extracting proteins from MDA-MB-231 cells
3. Performing the Lowry protein assay to quantify extracted proteins
4. Preparing different dilutions of bovine serum albumin (BSA) to be used as
standards
5. Constructing a standard curve of absorbance vs. concentration
6. Calculating the concentration of extracted proteins, using the obtained standard
curve

C. Introduction:
The extraction of biomolecules, including DNA, RNA and proteins, is the most
crucial method used in molecular biology. Biomolecules can be isolated from any
biological material, such as living or conserved tissues, cells, viral particles or other
samples, for analytical or preparative purposes.
Proteins can be extracted by a few methods such as detergent lysis, shearing forces
and rapid changes in pressure, which aim to weaken and break the membranes
surrounding the cell to release proteins. Detergents release soluble proteins by lysing
cells, and are also known to solubilize membrane proteins by mimicking the
phospholipid bilayer.
A number of factors should be considered when handling proteins. Normally, protein
extraction is performed at a very low temperature (4°C), as proteins are easily
denatured once they are released from the cells. Buffer condition is one of the major
factors that need to be controlled. Specific buffer conditions are recommended to be
maintained because of the sensitivity of proteins toward environmental changes (pH
and ionic strength). The pH of cell lysis buffers should be kept within the
physiological range (7.2-7.4) in order to prevent protein denaturation. The ionic
strength is another important buffer condition, as protein solubility is known to be
affected by salt concentration. Protease inhibitors are essential components of most
cell lysis and protein extraction procedures. These inhibitors block or inactivate
endogenous proteolytic enzymes that are released from subcellular compartments
during cells lysis and would otherwise degrade proteins of interest. Processing
samples at cold temperatures would also help reduce the activity of proteases.
Reducing agents may be added to the buffer for protein extraction to prevent the loss
of activity of proteins or enzymes caused by oxidization. Storage of proteins is
important as the half-life of protein is commonly dependent on the storage
temperature.
Protein purification is required to determine its unique characteristics, including size,
charge, shape and function. Several methods are commonly used in protein
purification. These include: ion-exchange chromatography, gel filtration
chromatography, affinity chromatography and gel electrophoresis.
The first step in protein purification is cell lysis. In order to purify and analyze protein
efficiently, they must be first released from cells in a soluble form. The plasma
membrane of mammalian cells, composed of phospholipids and proteins, can be
easily disrupted. In contrast, protein extraction from fungi and bacteria appears to be
more challenging due to the presence of a stable cell wall.
Ion-exchange chromatography, also referred to as ion chromatography, separates
proteins based on their net charge, which depends on the composition of the mobile
phase, using resin matrices that are modified with either positively-charged or
negatively-charged chemical groups. Most proteins have an overall negative or
positive charge at a given pH, depending on their isoelectric point (pI), the pH at
which they have no net electrical charge, which makes it possible for them to interact
with an oppositely-charged chromatographic matrix. At a pH below their pI, proteins
carry a net positive charge; above their pI, they carry a net negative charge. Thus,
proteins will bind to a cation exchanger (negatively-charged) in cation exchange
chromatography at a pH below their pI, and to an anion exchanger (positively-
charged) in anion exchange chromatography at a pH above their pI.
Proteins that interact weakly with the resins, for example a weak positively-charged
protein passed over resin modified with a negatively-charged group, are eluted out in
a low-salt buffer. On the other hand, proteins that interact strongly require more salt to
be eluted. Proteins with very similar charge characteristics can be separated into
different fractions as they are eluted from the column by increasing the concentration
of salt in elution buffer.
Gel-filtration chromatography, also referred to as size-exclusion or gel permeation
chromatography, separates proteins according to their size. It is a process in which
large molecules pass through the column faster than small molecules. Smaller proteins
are able to enter the tiny pores of the matrix and are, therefore, trapped and removed
from the flow of the mobile phase. The average residence (or retention) time in the
pores depends upon the effective size of the analyte molecules. On the other hand,
proteins that are larger than the average pore size of the packing are excluded, as they
cannot get into the pores, and thus suffer essentially no retention; such species are the
first to be eluted.
Affinity chromatography depends on a highly specific interaction between the target
protein (substrate, antigen, receptor, carrier protein ...) and the solid phase, typically a
gel matrix, to affect separation from contaminants. It consists of the same steps as ion-
exchange chromatography; however, it enables the purification of a protein on the
basis of its biological function or individual chemical structure. A biospecific ligand
(enzyme, antibody, hormone …) is covalently linked to a chromatography matrix.
Proteins that have a high affinity towards a ligand will bind to the column matrix, and
will thus become trapped, while other proteins will pass through the column. The
bound proteins will be eluted out from the column by a solution containing a high
concentration of the soluble form of the ligand. The binding between the ligand and
target protein molecules must be reversible to allow the proteins to be removed in an
active form.
Gel electrophoresis is a method used to separate proteins according to their size,
shape, charge and pI. Proteins are commonly separated using polyacrylamide gel
electrophoresis (PAGE). Several forms of PAGE exist and can provide different types
of information about proteins.
Non-denaturing (or native) PAGE separates proteins according to the net charge, size
and shape of their native structure. The proteins are driven by an applied current
through a gel matrix. Electrophoretic migration occurs because most proteins carry a
net negative charge in alkaline running buffers. The higher is the negative charge
density (charges per molecule mass), the faster a protein will migrate. In addition, the
pore size of polyacrylamide gel plays a role as a molecular sieve to separate different
sizes of proteins. The larger is the protein, the slower it migrates as it becomes more
entangled in the gel due to a larger frictional force of the gel matrix. Shape is also one
of the factors that affect separation. Compact globular proteins move faster than
elongated fibrous proteins of comparable molecular mass.
Denaturing and reducing sodium dodecyl sulfate (SDS)-PAGE, the most widely used
electrophoresis technique, separates proteins primarily by mass. SDS-PAGE will be
discussed in details in next week's lab session.
Two-dimensional (2D)-PAGE separates proteins by pI in the first dimension and by
mass in the second direction.
Once separated by electrophoresis, proteins can be detected in a gel with various
stains, transferred onto a membrane for detection by Western blotting or excised and
extracted for analysis by mass spectrometry.
Protein quantitation is often necessary before processing protein samples for
separation, isolation and analysis by chromatographic, electrophoretic and
immunochemical methods.
The measurement most commonly used in protein assays is absorbance of light.
Beer’s law states that if a solute absorbs light of a particular wavelength, the
absorbance is directly proportional to the concentration of that solute in solution.
Often the solute by itself does not absorb light. Therefore, one or more reagents must
be used to produce colored compounds in proportion to the concentration of an
unknown.
Colorimetric assays use standard curves created by measuring the absorbance of
solutions of known protein concentration, known as standards, in order to determine
the concentration of proteins in unknown samples. Several colorimetric methods are
available for determining the total protein content of a sample. These include dye
binding assays (Bradford) and copper ion based assays (Lowry and BCA).
Coomassie G-250 exists in multiple forms. As part of the Bradford solution, the dye
exists in its cationic state and takes on a reddish-brown color. The peak absorption of
the dye in this state is at 470 nm. When the dye binds to and interacts with amino
acids, the dye is converted to a stable unprotonated blue form, and the absorption
maximum shifts from 470 nm to 595 nm. This stable blue form of the dye is easily
observed and quantified in a spectrophotometer. There is a correlation to the amount
of blue color and the amount of proteins in the sample. The more proteins exist, the
more intense is the blue color.
Copper ion based protein assays involve two redox reactions. The protein solution is
first mixed with an alkaline solution of copper salt. Under alkaline conditions, cupric
ions (Cu2+) chelate with the peptide bonds resulting in reduction of cupric (Cu2+) to
cuprous ions (Cu+). If the alkaline copper is in excess over the amount of peptide
bonds, some of the cupric ions (Cu2+) will remain unbound to the peptide bonds and
are available for detection. Protein assays based on copper ions can be divided into
two groups: assays that detect reduced cuprous ions (Cu+) and assays that detect the
unbound cupric ions (Cu2+).
The cuprous ions are detected either with bicinchoninic acid (BCA) or Folin reagent
(a mixture of phosphomolybdic and phosphotungstic acid), as in the protein assays
based on the Lowry method. Reduction of Folin reagent by cuprous ions (Cu+)
produces a blue form that can be quantified by reading the absorbance at 750 nm. The
amount of color produced is proportional to the amount of proteins. The reaction of
cuprous ions (Cu+) with the BCA and color production is similar to that of Folin
reagent.
In the assays based on the detection of unbound cupric ions (Cu2+), the protein
solution is mixed with an amount of alkaline copper that is in excess over the amount
of peptide bonds. The unchelated cupric ions (Cu2+) react with and are detected by a
color-producing reagent. The amount of color produced is inversely proportional to
the amount of proteins.
By using a dilution series of known protein concentrations, one can generate a
spectrophotometric standard curve of absorbance vs. concentration. This curve can
then be used to estimate the quantity of proteins in an unknown sample, based upon
the intensity of color (absorbance).

D. Experimental Procedure:
In this experiment, you will extract proteins from MDA-MB-231 cells, and you will
subsequently perform the Lowry assay for protein quantification. You will prepare
different dilutions of BSA to be used as standards. You will then read the absorbance
of samples and standards using a spectrophotometer, construct a standard curve of
absorbance vs. concentration and use this curve in order to calculate the concentration
of proteins in your samples.
1. Protein Extraction:

 Wash the tissue culture plate twice with 5 ml of cold PBS.

 Totally remove PBS.
 Add 300 µl of lysis buffer to the tissue culture plate.
 Using a cell scraper, scrape well in order to collect cells.
 Transfer into a labeled microcentrifuge tube.
 Shear cells using 1 ml tuberculin syringe having at least 21 G needle.
 Centrifuge at 15,000 g for 30 minutes at 4 °C.
 Transfer the supernatant (contains proteins) into a new microcentrifuge tube
labeled with the sample name, cell nomenclature, date and the group's name
initials, and keep on ice.
 Store at -80 °C for long storage and -20 °C for close use.

2. Protein Quantification:

 Plug in and turn on the spectrophotometer.

 Set the wavelength to 750 nm, and allow the spectrophotometer to warm up for 15
minutes. Meanwhile, do the following:
 Label eight microcentrifuge tubes with the following labels: blank, 1, 2, 3, 4, 5, A
and B. Tubes 1-5 will be used for preparation of BSA standards. Tubes A and B
will be used to dilute your protein sample by a dilution factor of 2 and 4,
respectively.
 Label disposable cuvettes with the following labels: blank, 1, 2, 3, 4, 5, A, B and
S (for the undiluted protein sample).
 Prepare BSA standards according to the table below. Use a fresh tip for each,
pipette up and down to mix and keep on ice.

Volume of Stock
Concentration of Volume of BSA Solution (1.5
Tube
BSA (mg/ml) Lysis Buffer mg/ml)

Blank 0 100 µl 0 µl

1 0.3 80 µl 20 µl

2 0.6 60 µl 40 µl

3 0.9 40 µl 60 µl

4 1.2 20 µl 80 µl

5 1.5 0 µl 100 µl

 Dilute your protein sample according to the table below. Use a fresh tip each time,
pipette up and down to mix and keep on ice.

Tube Dilution Factor Volume of Lysis Buffer Volume of Protein Sample

A 2 50 µl 50 µl

B 4 75 µl 25 µl

 Pipet 25 µl of the blank, standards (tubes 1-5) and samples (tubes A, B and the
undiluted protein sample) into the appropriate cuvette, using a fresh tip each time.
 Add 125 µl of reagent A' (alkaline copper tartrate solution) into each cuvette.
 Pipette up and down to mix, using a fresh tip for each.
 Add 1 ml of reagent B (Folin reagent) into each cuvette.
 Pipette up and down to mix, using a fresh tip for each.
 Incubate at room temperature for 15 minutes. The absorbance will be stable for at
least 1 hour.
 Visually, estimate the protein concentration of your sample and its dilutions by
referring to the standards.
 Place the cuvette labeled "blank" into the spectrophotometer. Press the "0
ABS/100%T" button to calibrate the spectrophotometer.
 Place each of the remaining cuvettes into the spectrophotometer and read the
absorbance at 750 nm. Record absorbance readings.
 Plot a standard curve of absorbance versus BSA concentration using Excel.
Display the R-squared (R2) value and the equation on the chart.
 Calculate the concentration of proteins in your sample and its dilutions (A and B).
 If the protein concentration of your sample does not fall within the range of the
prepared standard concentrations, calculate the concentration of proteins in your
sample, using data from A or B, such that their concentrations fall within the
range of standard concentrations.

E. Lab Report:
Refer to the rubrics I, II, III, IV, VI and VII.