MCF-7 cells were cultured in Dulbecco's medium with 10% fetal bovine serum. Cells were washed with PBS then lysed on ice in a modified RIPA buffer with protease and phosphatase inhibitors (0.1 mM AEBSF HCl, 0.08 µM Aprotinin, 5 µM Bestatin, 1.5 µM E-64, 2 µM Leupeptin Hemisulfate, 1 µM Pepstatin A. Phosphatase inhibitors 1 mM NaF and 1 mM Na3VO4.)
Cell debris was removed by centrifugation. Protein concentration was determined with modified Lowry (BCA) assay. Protein concentration was adjusted to 2 mg/ml and then an equal volume of 2X SDS-PAGE sample buffer added.
Does not contain dissociating agents and has not been heated.

Whole cell lysate is supplied in 1X SDS sample buffer (62.5 mM Tris HCl, 2% SDS, 10% Glycerol and 0.005% bromophenol blue, pH 6.8, 10% glycerol) without dissociating agents (2-mercaptoethanol or dithiothreitol)

Store at -80oC for up to 3 months.  For continuous use, prepare working aliquots and store at  -80°C.  Avoid repeated freeze-thaw cycles.

Heat lysate to 95°C for 5 minutes and cool rapidly.
If dissociating conditions are desired, add reducing agent prior to heating.