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Western blotting (WB) is a widely practiced analytical technique to detect target proteins using antigen-specific antibodies. The technique involves three major processes: separation by size (Gel electrophoresis), transfer to solid support (Blotting), and marking target protein to visualize (Probing).
Gel electrophoresis is used to separate sample protein based on polypeptide length. The separated protein is transferred onto a nitrocellulose (NC) membrane where it is probed with primary antibody specific to target protein antigen. Typically, the membrane is incubated with antibody against the antigen of interest followed by a secondary antibody and detection.
| Reagents | Instruments and Consumables |
| Loading Buffer | Eppendorf Tube |
| Running Buffer | Eppendoff |
| Transfer Buffer | Centrifuge |
| Washing Buffer (PBST) | Incubation Plate |
| Blocking Buffer | Oscillating Table |
| Chemiluminescent Substrate | Electrophresis Apparatus |
| Protein Maker | Transfer Tank |
| Primary Antibody | Imager |
| Secondary Antibody-HRP |
Sample preparation
1. Measure the concentration of protein, determine the volume of protein and PBS buffer.
2. Take 16 μl sample and add 4 μl 5x loading buffer. Mix well.
3. Boil the samples for 5 min at 100°C.
4. Centrifuge at 5000 rpm in a microcentrifuge for 1 min.
Protein Separation
5. Load 10 μl sample into the wells of SDS-PAGE gel, along with molecular weight maker.
6. Run the gel at 80 V in stacking gel, about 30 min.
7. Increase the voltage to 100 V in separating gel, about 90 min.
8. Finish the run until the dye front reaches the bottom of the gel.
Protein Transfer
9. Cut NC membrane based on the gel size and wet it in methanol.
10. Assemble the transfer sandwich and make sure no air bubbles between the gel and NC membrane.
11. Place the cassette in the transfer tank, which maintains 4°C in the ice-water bath.
12. Transfer for 60 min at a constant current of 100 V.
Antibody Incubation
13. Block the membrane with 10% skim milk for 60 min at room temperature.
14. Add primary antibody in 5% skim milk and incubate overnight in 4°C on a shaker.
15. Rinse the membrane 3 times with PBST, 8 min at a time.
16. Incubate an HRP-conjugated secondary antibody for 60 min at room temperature.
17. Rinse the membrane 3 times with PBST, 8 min at a time.
Imager and Data Analysis
18. Prepare and apply chemiluminescent substrate (Solution A and B) to the membrane.
19. Capture the chemiluminescent signals using a camera-based imager.
20. Use image analysis software to read the band intensity of the interested proteins.