| Scientific Background: | PC-12 cells were maintained in Ham's F12K medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 82.5%; horse serum, 15%; fetal bovine serum, 2.5%. 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. 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. |
