Lysis of Mammalian Cells

Buffers

RIPA buffer	1x PBS 1% Nonidet P40 0.5% sodium deoxycholate 0.1% SDS

	Before use add the following: 10ug/ml PMSF (from a stock of 10mg/ml PMSF in isopropanol) 30ul/ml Aprotinin (Sigma #A6279) 10ul/ml 100mM sodium orthovanadate (store @-20C) OR Protease, and Phosphatase I and II Inhibitor Cocktails 1:100 (Sigma # P8340, P2850, and P5726)

PTY buffer		per 1000ml
	50 mM HEPES 50 mM NaCl 5 mM EDTA 1% Triton X-100 50 mM NaF 10 mM Na4P2O7	11.9 g 2.9 g 1.86 g 10 ml of 100% 2.1 g 4.46 g of the decahydrate salt
		pH to 7.4, filter through 0.45um, store at 4C
	Before use add	Protease, and Phosphatase I and II Inhibitor Cocktails 1:100 (Sigma # P8340, P2850, and P5726)
	Reference	O'Neill G.M. and Golemis E.A. Mol. Cell Biol. 2001 21(15):5094-108

Lysis protocol

Aspirate media from cells.
Wash with cold PBS, aspirate, place plate on ice.
Drain tissue culture plate of excess PBS.
Add lysis buffer, approximately 100-200 ul per 30 cm2 surface of culture plate.
(e.g. for 35 mm dish add 50-100 ul, for a 60 mm dish add 100-200 ul, for a 10 cm dish 300-500 ul)
Swirl dish, so that lysis buffer covers the entire surface.
Incubate on ice for 10 min.
Scrape cells with a cell scraper or cell lifter, collect lysate on one side of dish and let drain.
Collect lysate in a 1.5 ml eppendorf tube.
Incubate on ice for 10 min.
Sonicate lysate, or pass through 25 gauge needle attached on a 1 ml syringe.
Incubate on ice for 10 min.
Spin at 14,000 rpm for 10 min at 4C.
Transfer lysate on a new 1.5 ml eppendorf tube, discard pellet.
Store lysate at -80C