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Western Blot

Authors: Alex Kuo and Peggie Cheung, updated date: , view: 245, Q&A: 0

Procedure

Take down/resuspend lysate in RIPA buffer

10 mLRIPA + 1 protease inhibitor cocktail + 100 ul PMSF

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

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