Direct-Blot™ Western Blotting Protocol
Place cells in a microcentrifuge tube and centrifuge to collect the cell pellet.
Lyse the cell pellet with 100 µl of lysis buffer on ice for 30 min (For 1 X 106 cells, lyse with 100 µlof lysis buffer).
Centrifuge at 14,000 rpm (16,000 x g) for 10 minutes at 4°C.
Transfer the supernatant to a new tube and discard the pellet.
Remove 20 µl of supernatant and mix with 20 µl of 2x sample buffer.
Boil for 5 min.
Cool at room temperature for 5 minutes.
Microcentrifuge for 5 minutes.
Load up to 40 µl of sample to each well of a 1.5 mm thick gel.
Set gel running conditions according to the manufacturer’s instructions.
Transfer the proteins to anitrocellulose or PVDF membrane with variable power settings according to the manufacturer’s instructions.
Remove the blotted membrane from the transfer apparatus and immediately place in blocking buffer consisting of 5% nonfat dry milk/TBS-T. Incubate the blot for 1 hour at room temperature, or overnight at 4°C with agitation.
Dilute the Direct-Blot™ antibody to the recommended concentration/dilution in 5% nonfat dry milk/TBS-T** (usually at a 1:1000-1:2000 dilution).
Place the membrane in the Direct-Blot™ antibody solution and incubate for 2 hours at room temperature, or overnight at 4°C with agitation.
Wash  for 5 minutes with Wash Buffer (TBS containing 0.1% Tween-20).
[wash 1/3] Wash  for 5 minutes with Wash Buffer (TBS containing 0.1% Tween-20).
[wash 2/3] Wash  for 5 minutes with Wash Buffer (TBS containing 0.1% Tween-20).
[wash 3/3] Incubate membrane (protein side up) with 10 ml of ECL (enhanced chemiluminescence substrate) for 1-2 minutes.
The final volume required is 0.125 ml/cm2.
Drain off the excess detection reagent, wrap up the blots, and gently smooth out any air bubbles.
Place the wrapped blots, protein side up, in an X-ray film cassette and expose to x-ray film.
Exposures can vary from 5 seconds to 60 minutes.
