Western Blotting

 WESTERN BLOTTING  

AIM: To learn the technique of western blotting for the detection of a specific protein.  

INTRODUCTION: Western blotting or protein immunoblotting is a very sensitive and analytical method that involves detection of specific protein in a complex mixture. Protein samples are first separated using SDS polyacrylamide gel electrophoresis (SDS-PAGE) followed by the immobilization of protein or nitrocellulose or PVDF membranes. The transfer of proteins from the gel to the membrane is done electrophoretically. The transferred protein is detected using specific primary antibody and secondary enzyme labelled antibody and substrate. This method utilizes the principle of antigen-antibody interaction for identification of specific antigens by monoclonal or polyclonal antibodies.  

PRINCIPLE: 

Western blotting or immunoblotting is a method used for identifying a specific protein in a complex mixture along with the determination of the molecular weight. Protein samples are first electrophoresed on SDS-PAGE. In this process proteins migrate through the gel and they are separated according to their size and charge. These separated proteins electrotransfer onto nitrocellulose/ PVDF membrane for further analysis. To detect the protein (antigen) blotted on the membrane it is incubated with an antibody (primary) specific for the protein of interest. The membrane is thin, incubated with a second antibody (secondary) which is specific for the first antibody. The secondary antibodies are covalently attached to an enzyme, Eg: alkaline phosphatase or horseradish peroxidase. These enzymes form a colored precipitate upon reacting with a chromogenic substrate. As a result a viable band can be seen on the membrane where the primary antibody is bound to the protein. This entire procedure can be divided into the following steps.  

SDS-PAGE: 

SDS-PAGE (Sodium dodecyl sulphate polyacrylamide gel electrophoresis) is a technique used in biochemistry, genetics and molecular biology to separate proteins according to their molecular weight. The electrophoretic mobility of proteins depends on their size. The purpose of SDSPAGE is to separate proteins according to 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 condition, the electrophoretic separation of proteins is determined by both size and charge of the molecules. If proteins are high molecular weight molecules, its porous gel to gel separated - polyacrylamide gel are those which provide anions for separation by size as they are porous.  

Western Blotting : 

 Immunoblotting or western blotting is the electro transfer of resolved proteins from the polyacrylamide gel to the nitrocellulose/ PVDF membrane in the presence of a specific buffer called transfer buffer. For this transfer procedure the gel is placed on the membrane and both of them are sandwiched between two filter papers.  

 

 

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 : 

 After electro transfer,  proteins bound to the membrane are detected immunologically. The process is known as Immunodetection or Immunoblotting. A suitable blocking reagent (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 (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  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 colored 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  : 

1. Glass wares: conical flask, measuring cylinder, beaker, petri- dish, staining tray. 

2. Reagents: Methanol, distilled water . 

3. Other requirements: Protein electrophoresis apparatus, Gel maker, Micro pipette tips, Microwave/Burner/hot plate.   

4. 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, Ammonium persulphate (APS),  Tetramethylethylenediamine (TEMED),  agarose,  protein sample,  10X transfer buffer, 10X wash buffer, primary antibody,  secondary antibody,  TMB/ H2O2,  Nitrocellulose membrane with filter paper.  

 PROCEDURE : 

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% Separation Gel - To prepare separating gel, add the components as follows:     30% Acrylamide-bisacrylamide Solution - 6 ml  

                   Distilled water - 3 ml  

                   2.5X Tris-SDS Buffer (pH 8.8) - 6 ml 

                   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 one 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 6.8) - 1.6 ml 

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 a 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 40µl of protein sample in a tube and add 8 µl of  5X  sample loading buffer to it . Boil the tube containing protein ladder. 

8. Load samples in alternative wells as follows : 

Lane 1 - Prestained protein ladder - 5 µl 

Lane 2 - Protein sample - 20 µl 

Lane 3 - 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 ,staining and 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 40 C. 

Day-2  Immunodetection  

1. Discard off the blocking buffer. 

2. Wash the membrane with 20ml of 1X wash buffer for 5min. Repeat the wash once. 

3. Immerse the membrane in 20ml of 1X  of 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 20ml of 1X Wash buffer for 5 min. Repeat the wash once. Discard the buffer each time. 

5. Immerse the blot in 20 µl of 1X Assay buffer. Add 2ml of HRP labeled secondary antibody. Mix gently for an hour and 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 20ml of 1X Wash buffer for 10 min. 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 min, within this time coloured band will appear. 

8. Remove the blot, wash the distilled water, discard and dry. 

9. Compare the SDS polyacrylamide gel with the developed membrame. 

OBSERVATION: 

1. On staining SDS- polyacrylamide gel, different proteins will appear as dark and blue bands against a light blue background. 

2. On Immunodetection a single blue band will be observed on the nitrocellulose membrane. 

RESULT: 

On electrophoresis of the bacterial lysate on the SDS-PAGE many bands indicating the different proteins present in the crude sample as seen. A predominant band among these is that of the GST antibody. However few light bacteria may be seen indicating the proteins with which anti- GST antibody cross reacts.