3T3 (heat shocked) , (cell lysate)

Formulation:	Frozen lysate.

Source/Host:	Mouse Fibroblast

Application:	Western Blot (control, 20mg) Optimal conditions must be determined individually for each application.

Long Term Storage:	-80°C

Miscellaneous/General:	3T3 cells were maintained in Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, 90%; bovine calf serum, 10%. 3T3 cells were heat shocked at 44°C for 2 hours and allowed to recover for five to eighteen hours at 37°C before harvesting. Cells were collected from media by centrifuging at 1200 rpm at 4°C in a Beckman GS-6R centrifuge for 7 minutes. Cells were washed twice in PBS and were lysed in M-PER Mammalian Protein Extraction Reagent (Pierce catalog# 78501) with a protease inhibitor cocktail. The cell lysate was adjusted to 2mg/mL in SDS PAGE sample buffer (40 mM Tris-HCl pH 6.8, 1% SDS, 50 mM DTT, 7.5% glycerol, 0.003% bromophenol blue) and heated for 10 min at 70°C.