|Chaps Cell Extract Buffer (10X)||5 ml|
|30X Reducing Agent (1.25 M DTT)||250 µl|
Treat cells by adding fresh media containing regulator for desired time.
Aspirate media from cultures; wash cells three times with PBS; aspirate. Scrape cells into PBS and spin down to pellet.
Lyse cells by adding Chaps Cell Extract Buffer (1 volume of cell pellet). Resuspend cells in the buffer, freeze and thaw three times, centrifuge lysate at 14,000 rpm. Keep the supernatant and discard the pelleted cell debris.
Add SDS Sample Buffer and heat sample to 95–100°C for 5 minutes; cool on ice.
Microcentrifuge for 5 minutes.
Load 5 µL onto SDS-PAGE gel (10 cm x 10 cm).
CHAPS Cell Extract Buffer (5mL) 1X concentration: 50 mM Pipes/HCl (pH 6.5), 2 mM EDTA, 0.1% Chaps, 20 µg/ml Leupeptin, 10 µg/ml Pepstatin A,10 µg/ml Aprotinin. Add DTT to 5mM.1.25 M (250X) DTT (0.25 mL).
Supplied as a 10X solution. Store at -20°C
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