Take down/resuspend
lysate in RIPA buffer
Sonicate 10 minutes
on HIGH
Centrifuge at 4
degrees full speed 5 min
Remove supernatant
Measure
concentration by Bradford
500 ul Reagent A +
10 ul Reagent S
20 ul in each tube +
2 ul of sample/control + 160 ul reagent B
Sit for 10 minutes
Read on Nanodrop Protein Lowry
Adjust concentration
with 5X SDS and RIPA
Boil for 5 minutes
Store samples at -20
Load samples onto
gel
Run 45 minutes @ 200
V
Transfer with 2
blotting pads onto PVDF membrane
Run 30 minutes @ 25
V
Block in 10% milk
for ~10 minutes
Primary antibody for
1 hour at RT or 4 degrees overnight
Wash 3x with TBST
Secondary antibody
for 30 min at RT
Wash 3x with TBST
For PBMCs: 1 million
cells to total volume 100 ul 1X SDS + RIPA