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 primary antibody to the recommended concentration / dilution in 5 % nonfat dry milk / TBS - T * * ( usually at 1µg / ml ) .
Place the membrane in the primary 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 the membrane for 30 minutes at room temperature with horseradish peroxidase ( HRP ) ‐conjugated secondary antibody , diluted to 1 : 1000 in 5 % nonfat dry milk / TBS‐T .
Wash for 10 minutes with TBS containing 0.1 % Tween‐20 .
( 1 / 4 ) .
Wash for 10 minutes with TBS containing 0.1 % Tween‐20 .
( 2 / 4 ) .
Wash for 10 minutes with TBS containing 0.1 % Tween‐20 .
( 3 / 4 ) .
Wash for 10 minutes with TBS containing 0.1 % Tween‐20 .
( 4 / 4 ) .
Wash once for 2 minutes with PBS .
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 .
