Western Blot Protocol, Principle, Result.

What is Western Blotting?

In molecular biology Western blotting is a rapid and sensitive assay for detection and characterization of proteins.  This technique exploits the inherent specificity of antigen-antibody interaction to identify specific antigens by polyclonal or monoclonal antibodies.

• Western blotting is also known as protein immunoblot because an antibody is used to specifically detect its antigen.
• The western blotting is completed in three steps such as separation of protein by size, transfer of protein to a solid support, and marking target protein using a primary and secondary antibody to visualize.
• At first the proteins of a given sample are separated by using SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
• These separated proteins are transferred or blotted onto a matrix generally known as nitrocellulose or PVDF membrane.
• After that the secondary antibodies are added to it which will bind with the specific primary antibodies. Now these secondary antibodies are visualized by using different methods such as staining, immunofluorescence, and radioactivity, allowing indirect detection of the specific target protein.

Aim

To learn the technique of Western Blotting for the detection of a specific protein. 

Western Blotting Principle

The western blotting technique is mainly used to identify a specific protein in a complex mixture along with the determination of its molecular weight. At first, the protein samples are electrophoresed on SDS-PAGE. During this time the proteins will migrate through the gel and they will be separated according to their size and charge.

These separated proteins are electrotransferred onto nitrocellulose/PVDF membrane for further analysis. To confirm the electrotransferred proteins or antigen are blotted on the membrane it is incubated with a primary antibody which is specific for the protein of interest. 

After that, the membrane is incubated with the secondary antibody which is specific for the first antibody. The secondary antibodies are covalently attached to an enzyme, e.g. alkaline phosphatase or horseradish peroxidase. These enzymes form a coloured precipitate upon reacting with a chromogenic substrate. 

As a result, a visible band can be seen on the membrane where the primary antibody is bound to the protein. 

Western Blot test Steps

The entire procedure can be divided into the following steps:

SDS-PAGE

• In biochemistry, genetics and molecular biology, the SDS-PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis are used to separate proteins according to their molecular weight. The electrophoretic mobility of proteins depends upon their size. 
• The main purpose of SDS-PAGE is to separate the proteins based on their size.
• As proteins are amphoteric compounds, their net charge can therefore be determined by the pH of the medium in which they are suspended. 
• Therefore, at a given pH and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of molecules. As proteins are high molecular weight molecules, it needs porous gels to get separated.
• Polyacrylamide gels are those which provide a means of separating proteins by size as they are porous.

Western blotting

• The resolved proteins are transferred from the polyacrylamide gel to the nitrocellulose/PVDF membrane in presence of a specific buffer termed transfer buffer.
• During the transfer procedure, the gel and membrane are placed between two filter papers like a sandwiched, while the gel will be at the top of the membrane.
• This set is placed between two sponge pads and then placed in a plastic cassette.
• The entire set is then placed inside a gel tank filled with cold transfer buffer. 
• The resolved proteins are transferred to the corresponding positions on the membrane after the electrotransfer. The protein of interest is immunodetected on the membrane. 

Immunodetection

• In Immunodetection or Immunoblotting, the proteins which are bound to the membrane after the completion of electrotransfer, are detected immunologically.
• A suitable blocking reagent such as non-fat dry milk/BSA is used to block the unoccupied sites on the membrane.
• Then the membrane is probed with a primary antibody specific to the protein of interest. The primary antibody binds to the protein or antigen and an antigen (Ag)-antibody (Ab) complex is formed on the membrane. 
• The membrane is washed to remove excess unbound primary antibody.
• It is then treated with an enzyme-labeled (Alkaline phosphatase/Horseradish peroxidase) secondary antibody which attaches to the primary antibody of the Ag-Ab complex.
• Finally, the membrane is incubated in a solution containing phosphatase or peroxidase substrate which results in a visible coloured band on the membrane where the Ag-Ab complex is formed.
•  As a result the molecular weight of the protein of interest can be determined.

Materials Required for Western Blot

• Acrylamide-Bisacrylamide Solution 30% (29:1) 
• 2.5X Tris-SDS Buffer (pH 8.8)
• 5X Tris-SDS Buffer (pH 6.8)
• Prestained Protein Ladder
• 5X Tris-Glycine-SDS Gel Running Buffer
• 5X Sample Loading Buffer
• Staining solution
• Destaining solution
• Ammonium persulphate (APS)
• Tetramethylethylenediamine (TEMED)
• Agarose
• Protein Sample
• Transfer Buffer
• Blocking Agent
• Diluent Buffer
• Assay Buffer
• Wash Buffer
• Primary antibody
• Secondary antibody
• TMB/H2O2
• Nitrocellulose membrane with filter paper
• Glass wares: Conical flask, Measuring cylinder, Beaker, Petri dish, staining tray
• Reagents: Methanol, Distilled water
• Other: Protein Electrophoresis apparatus, Blotting Apparatus, Power pack, Gel rocker, Micropipettes, Tips, Microwave/Burner/Hotplate 

Preparation of buffer and solution

• Preparation of 10% APS Solution: Dissolve 0.15 g of Ammonium persulphate in distilled water to make a final volume of 1.5 ml. Store at 2-8o C. Use within 3 months.
• Preparation of 1X Tris-Glycine-SDS Gel Running Buffer: To prepare 500 ml of 1X Tris-Glycine-SDS Gel Running buffer, take 100 ml of 5X Tris-Glycine-SDS Gel Running Buffer and add 400 ml sterile distilled water*. Store at 2-8o C. Mix well before use. The 1X Tris-Glycine-SDS Gel Running Buffer can be reused 4-5 times.
• Preparation of 1X Assay Buffer: To prepare 50 ml of 1X Assay Buffer, take 5 ml of 10X Assay Buffer and add 45 ml of sterile distilled water*
• Preparation of 1X Transfer Buffer: To prepare 1000 ml of 1X Transfer Buffer, take 100 ml of 10X Transfer Buffer, add 200 ml of methanol and 700 ml of sterile distilled water*. Store at 2-8o C. Mix well before use.
• Preparation of Blocking Buffer: To prepare 20 ml of Blocking Buffer, take 0.2 g of Blocking Agent and add 20 ml of Diluent Buffer.
• Preparation of 1X Wash Buffer: To prepare 1000 ml of 1X Wash Buffer, take 100 ml of 10X Wash Buffer and add 900 ml of sterile distilled water*.

Western Blot Protocol

Day 1: SDS- PAGE

1. Assemble the electrophoresis unit such that the glass plates are clamped to the unit along with the spacers placed in-between them at two vertical edges.
2. Prepare 1% agarose (0.05g in 5ml of distilled water). Boil to dissolve the agarose and pour a thin horizontal layer at the lower edge of the plates to seal the assembly. Let it solidify by allowing it to cool down for 5-10 minutes
3. Preparation of 12% Separating Gel- To prepare separating gel, add the components as follows:

30% Acrylamide-bisacrylamide Solution	6ml
Distilled water*	3ml
2.5X Tris-SDS Buffer (pH 8.8)	6ml
10% APS Solution	125 μl
TEMED	18 μl

Pour the gel in-between the plates and allow it to solidify for an hour. Immediately after the gel is poured, add distilled water to level the gel. 

4. After an hour pour off the water by inverting the casting assembly.
5. Preparation of 5% Stacking Gel- To prepare stacking gel, add the components as follows:

30% Acrylamide-bisacrylamide Solution	1.3 ml
Distilled water*	5.1 ml
5X Tris-SDS Buffer (pH 8.8)	1.6ml
10% APS Solution	75 μl
TEMED	10 μl

After addition of TEMED gently mix all the components by swirling the beaker. Pour the stacking gel on top of the separating gel and immediately place the comb avoiding air bubbles. Allow it to solidify for 30 minutes.

Note: Acrylamide is a potential neurotoxin and should be treated with great care. Always wear an face mask and use gloves. 

6. Pour 1X Tris-Glycine-SDS Gel Running Buffer in the unit such that the buffer connects the two electrodes, and hence completes the flow of current. Remove the comb from the Stacking Gel carefully.
7. Sample Preparation: Take required amount of protein sample in a tube and boil the tube containing protein sample at 100o C in a boiling water bath. Do not boil the tube containing Prestained Protein Ladder.
8. Load samples in alternative wells as follows: Lane 1: Prestained Protein Ladder – 5 μl; Lane 3: Protein Sample – 20 μl; Lane 5: Protein Sample – 20 μl
9. Connect the power cord to the electrophoretic power supply according to the conventions: Red-Anode and Black- Cathode. Electrophorese at 120 volts and 90 mA until dye front reaches 0.5 cm above the sealing gel.
10. Carefully remove the gel from in-between the plates using spatula into the plastic tray containing distilled water. Wash the gel for 1 minute. Discard the water & proceed for blotting and staining destaining procedure.
11. To the gel pieces of lane no. 1 and 3 add 20 ml of water and proceed for staining destaining procedure.
12. Cut the Gel along lane no. 4. Transfer lane no. 5 i.e. protein sample in 10 ml of cold Transfer buffer. Incubate at Room Temperature for 10 minutes and proceed with electroblotting.

Staining and Destaining of Gel

1. After removing water, add 50 ml of Staining Solution in the tray containing gel, till the bands are visible. Sometimes the gel may have to be kept overnight in the staining solution for visualization of the bands.
2. Remove gel from the Staining Solution. The Staining Solution can be re-used 2-3 times.
3. Wash the gel by rinsing with distilled water till a considerable amount of stain leaches out from the gel. Keep changing the distilled water for 3-4 times.
4. Add 50 ml of Destaining Solution to the gel. Destaining should be carried out with constant moderate shaking.
5. Continue destaining till clear, distinct bands are observed.
6. Remove gel from the Destaining Solution. The Destaining Solution can be re-used 2-3 times.

Electroblotting

1. Assemble the gel with nitrocellulose membrane and filter papers.This blotting sandwich is placed within the blotting cassette. Try to avoid air bubble between gel and nitrocellulose membrane by rolling a glass tube on the membrane.

Note: Take out the transparent sheets carefully while using the nitrocellulose membrane.

2. Insert this cassette into the gel transfer apparatus filled with cold transfer buffer and then connect the transfer unit to power supply as per conventions.
3. Electrophoreses the sample at 150V, 300 mA for 2 hours for blotting.
4. Remove the nitrocellulose membrane after electrophoresis from the blotting cassette and place the membrane (with protein side up) in 20 ml of 1X Blocking Buffer taken in petri dish.
5. Keep it overnight at 40C.

Day 2: Immunodetection:

1. Discard off the blocking buffer.
2. Wash the membrane with 20 ml of 1X Wash Buffer for 5 minutes. Repeat the wash once.
3. Immerse the membrane in 20 ml of 1X Assay Buffer. Add 4 μl of primary antibody solution and mix gently for an hour on a gel rocker. After that discard the primary antibody solution.
4. Wash the blot with 20 ml of 1X Wash Buffer for 5 minutes. Repeat the wash once. Discard the buffer each time.
5. Immerse the blot in 20 ml of 1X Assay Buffer. Add 2 μl of HRP labeled secondary antibody. Mix gently for an hour. Discard the HRP labeled antibody solution.
6. Do a quick washing of the blot with 20 ml of 1X Wash Buffer. Wash the blot with 20 ml of 1X Wash Buffer for 10 minutes. Repeat the wash. Discard the buffer each time.
7. Immerse the washed blot in 3 ml of TMB/H2O2 (substrate) solution, mix gently for 5-10 minutes, within this time coloured band will appear.
8. Remove the blot; wash with distilled water, discard and dry.
9. Compare the SDS-Polyacrylamide gel with the developed membrane.

Observation and Result

• On staining SDS-Polyacrylamide gel, different proteins will appear as dark blue bands against a light blue backgroud.
• On immunodetection, a single blue band will be observed on NC membrane.

• Lane 1: Prestained Protein Ladder
• Lane 3: Protein Sample
• Lane 4: Immunodetection on the blotted membrane

Interpretation:

After staining and destaining of the gel several bands appear in the sample which is a crude bacterial cell lysate. After performing the Western blotting procedure a thick band can be seen on the nitrocellulose membrane which corresponds to the GST protein which is detected by anti-GST antibody. The molecular weight of GST protein is 26 kD and the position of the band corresponds to the protein size.

Application of western blotting

• Help to determine the size and amount of protein in a supplied sample.
• Used in diagnosis of different disease.
• Help to detect the  HIV antibody in patient’s serum.
• Detect defective proteins.
• Used  as the definitive test for variant Creutzfeldt-Jakob Disease, Bovine spongiform encephalopathy.
• Used in diagnosis of tularemia.
• Used as a confirmatory test for Hepatitis B infection and HSV-2 (Herpes Type 2) infection.

References

• https://www.bosterbio.com/protocol-and-troubleshooting/western-blot-principle
• https://himedialabs.com/td/hti009.pdf
• http://www.ispybio.com/search/protocols/WesternBlotting.pdf
• https://en.wikipedia.org/wiki/Western_blot
• https://www.mybiosource.com/learn/westernblotting
• https://www.antibodies.com/western-blotting