Western Blotting Protocol
Sample preparation:
1. Place cells in a microcentrifuge tube and centrifuge to collect cell pellet.
2. Lyse the cell pellet with 100 μl lysis buffer on ice for 30 min (For 1 X 106 cells,
lyse with 100 μl lysis buffer).
3. Centrifuge at 14,000 rpm (16,000 x g) for 10 min at 4°C.
4. Transfer the supernatant to a new tube and discard the pellet. Remove 20 μl
supernatant and mix with 20 μl of 2x sample buffer.
5. Boil for 5 min. Cool at RT for 5 min. Microcentrifuge for 5 min.
6. Load up 40 μl of sample to each well of a 1.5 mm thick gel*.
7. Set gel running conditions according to the manufacturer’s instructions.
Transfer the proteins to a nitrocellulose or PVDF membrane with variable
power settings according to the manufacturer’s instructions.
*Guidelines for choosing gel percentages are based on protein size to be
detected: 4-5% gel, >200 kD; 7.5% gel, 120-200 kD; 8-10% gel, 40-120 kD; 13%
gel, 15-40 kD; 15% gel, < 20 kD.
Membrane Blocking:
1. Remove the blotted membrane from the transfer apparatus and immediately
place in blocking buffer consisting of 5% nonfat dry milk/TBS-T**.
2. Incubate the blot for 1 hr at room temperature, or overnight at 4°C with
agitation.
Antibody Incubation:
1. Dilute the primary antibody to the recommended concentration/dilution
in 5% nonfat dry milk/TBS-T** (usually at 1 μg/ml). Place the membrane in
the primary antibody solution and incubate 2 hrs at room temperature, or
overnight at 4°C with agitation.
2. Wash three times for 5 min each with Wash buffer (TBS containing 0.1%
Tween-20).
3. Incubate the membrane for 30 min at room temperature with horseradish
peroxidase (HRP)-conjugated secondary antibody, diluted to 1:1000 in 5%
nonfat dry milk/ TBS-T**.
4. Wash 4 times for 10 min each with TBS containing 0.1% Tween-20, and once
for 2 min with PBS.
Protein Detection:
1. Incubate membrane (protein side up) with 10 ml ECL (enhanced chemiluminescence
substrate) for 1-2 min. The final volume required is 0.125 ml/cm2.
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