EXPERIMENT

AIM: To perform Western- Blot analysis to check specificity of given protein sample

REQUIREMENTS

Materials- Sponge pad, filter papers, transfer apparatus, SDS-PAGE gel with separated proteins, rocker-
shaker, measuring cylinders, etc.

Solutions-

A) Resolving Gel

1.5MTris 3.8 ml
30% acrylamide 5 ml
10% SDS 150 ml
10% APS 150 ml
TEMED 10 ml
Distilled water 5.9 ml

B) Stacking Gel

1MTris (6.8 pH) 680 ml

30% acrylamide 1 ml
10% SDS 50 ml
10% APS 50 ml
TEMED 10 ml
Distilled water 3.3 ml

C) Transfer Buffer (1 litre)

Tris base 5.82g

Glycine 2.93g
10% SDS 3.75 ml
Distilled water 800ml
Methanol 200ml

D) 1X PBST (Phosphate Buffered Saline Tween-20)

NaCl 8g
KCl 0.2g
Na2HPO4 1.44g
KH2PO4 0.24g
Tween-20 2ml
Dissolve in 800ml distilled water. Then make up the volume to 1L.
THEORY

Western blotting, also known as immunoblotting, is a well-established and widely used technique for the
detection and analysis of proteins. The method is based on building an antibody: protein complex via
specific binding of antibodies to proteins immobilized on a membrane and detecting the bound antibody
with one of several detection methods.
Western Blotting or immunoblotting allows determining the presence of a specific amino acid sequence in
a sample after separation on SDS-PAGE(sodium dodecyl sulphate polyacrylamide gel electrophoresis). The
term Western blotting is used after a similar term ‘Southern blotting’, which was invented by and named
after E. M. Southern.

PRINCIPLE

Western blotting is the transfer of proteins from the SDS- PAGE gel to a solid supporting membrane. There
are two types of blotting apparatus used to transfer proteins to solid supports; these facilitate either wet
transfer (tank blotting) or semidry transfer. Both give good result.

Electrophoresis - The separation of macromolecules in an electric field. Common method for separating
proteins by electrophoresis uses a discontinuous polyacrylamide gel as a support medium and sodium
dodecyl sulphate (SDS) to denature the proteins. The method is called sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE).

SDS (also called lauryl sulphate) is an anionic detergent, meaning that when dissolved its molecules have a
net negative charge within a wide pH range. A polypeptide chain binds amounts of SDS in proportion to its
relative molecular mass. The negative charges on SDS destroy most of the complex structure of proteins,
and are strongly attracted toward an anode (positively-charged electrode) in an electric field.

Polyacrylamide gels - restrain larger molecules from migrating as fast as smaller molecules. Because the
charge-to-mass ratio is nearly the same among SDS-denatured polypeptides, the final separation of
proteins is dependent almost entirely on the differences in relative molecular mass of polypeptides. In a
gel of uniform density, the relative migration distance of a protein (Rf, the f as a subscript) is negatively
proportional to the log of its mass. If proteins of known mass are run simultaneously with the unknowns,
the relationship between Rf and mass can be plotted, and the masses of unknown proteins estimated.

The SDS-PAGE is a separating gel topped by stacking gel and secured in an electrophoresis apparatus.
Sample proteins are solubilised by boiling in the presence of SDS and equal amount of the protein in
solution are loaded into a gel lane, and the individual proteins separated electrophoretically. 2-
mercaptoethanol and dithiothreitol are added to reduce disulfide bonds.

A protein sample is subjected to polyacrylamide gel electrophoresis. After this the gel is placed over a
sheet of nitrocellulose and the protein in the gel is electrophoretically transferred to the nitrocellulose. The
nitrocellulose is then soaked in blocking buffer (3% skimmed milk solution) to "block" the non-specific
binding of proteins. The nitrocellulose is then incubated with the specific antibody for the protein of
interest. The nitrocellulose is then incubated with a second antibody, which is specific for the first
antibody. The second antibody will typically have a covalently attached enzyme which, when provided with
a chromogenic substrate, will cause a colour reaction. Molecular weight and amount of the desired
protein can be characterized from a complex mixture of other proteins by western blotting.
Advantages of western blotting-

1. Sensitivity -western blot can detect as little as 0.1 nano grams of protein in a sample, the technique
can theoretically serve as an effective early diagnostic tool, sensing even the slightest immunogenic
response from a virus or bacteria in a patient sample. Greater sensitivity means that fewer
antibodies are needed for testing, which cuts down laboratory costs significantly.
2. Specificity - gel electrophoresis sorts a sample into proteins of different size, charge, and
conformation. This process is a huge step towards detection, as bands formed in the gel already
give clues about the size of the protein or polypeptide of interest. The specificity of the antibody-
antigen interaction serves as the second big factor. Because specific antibodies show affinity for
specific proteins, the process can selectively detect a target protein even in a mixture of 300,000
different proteins.

Disadvantages of western blotting-

1. Prone to False or Subjective Results- in spite of its sensitivity and specificity, a western blot can still
produce erroneous results. A false-positive result when an antibody reacts with a non-intended
protein, which is what frequently happens when a patient being tested for HIV happens to have
tuberculosis or several parasitic infections. A false-negative, on the other hand, can easily result if
larger proteins are not given sufficient time to transfer properly to the membrane. Improper
blotting and processing often produce skewed, faded, or even multiple bands, making test results
subject to the interpretation of the technician.
2. High Cost - equipment’s required for detection and imaging -- chemiluminescent, fluorescent,
radioactive, or laser detection systems, can be too expensive for the average microbiology unit.

PROCEDURE

1. 6 filter paper sheets that fit the measurement of the gel and one PVDF membrane with the same
dimensions are taken.
2. A transfer sandwich is created in transfer buffer as follows: sponge, 3 filter papers, PVDF, gel, 3
filter papers and sponge.
3. The sandwich is relocated to the transfer apparatus, which is placed on ice to maintain 4̊C.
Transfer buffer is added to the apparatus and it is ensured that the PVDF membrane is between the
gel and a positive electrode.
4. The transfer is done for 45 minutes at 125mA at 4̊C.
5. The membrane is then blocked with 3% BSA in PBS (overnight incubation).
6. The membrane is then washed with 1X PBST thrice for 5 minutes each on a rocker shaker.
7. Primary antibody (Anti-GST in mouse) is added to the membrane and incubation is done at RT for 2
hours or at 4̊C for overnight.
8. The membrane is then again washed with 1X PBST thrice for 5 minutes each on a rocker shaker.
9. Secondary antibody (Anti-primary antibody in mouse) is added to the membrane and incubation is
done at 4̊C for 45 minutes each on a rocker shaker.
10. The membrane is then again washed with 1X PBST thrice for 5 minutes each on a rocker shaker.
11. The blot is then developed by using ECL (enhanced chemiluminescence) solution.
Result:
Western blotting was carried out successfully from SDS- PAGE. The protein could be visualized as bands
on blotting membrane.

Discussion:

Western blotting is one of the most common laboratory technique since it is accomplished rapidly,
using simple equipment and inexpensive reagents.
Well 1
Contain flow through which contained unbound as well as weakly bound proteins. Therefore, large
concentration of proteins was present in flow through corresponding to distinctive bands on blotting
membrane.
Well 2, 3, 4
Since all the unbound and weakly bound proteins come out in flow through, no protein bands were
found in this case.
Well 5, 6, 7
Elution buffer contain the bound proteins which were maximum in elution 1 buffer followed by elution
2, elution 3. Therefore, dark bands were found in elution 1 and faint bands were found for elution 2
and elution 3.

Precautions:
1. Do not touch the PVDF membrane with hands. Wear gloves while handling the gel and PVDF
membrane.
2. Ensure there are no air bubbles between the gel and PVDF membrane, and squeeze out extra
liquid.
3. The running time of transfer should be proportional to the thickness of the gel.
4. Extreme care should be taken while placing the membrane on the gel.
5. The transfer apparatus should not be disturbed while the transfer is taking place.
6. The sandwich should be made in transfer buffer ensuring the sponge pads, filter papers, membrane
and gel are all wet.