Multi-purpose Whole Cell Lysates produced by Rockland Immunochemicals are derived from tissues and cell lines using highly refined extraction protocols to ensure exceptionally high quality, protein integrity and lot-to-lot reproducibility. All extracts are tested by SDS-PAGE using 4-20% gradient gels. Tissues were washed exhaustively with PBS to remove blood and other debris. A lysate was prepared by homogenizing the tissue and washing the cells in cold PBS. Washed cells were incubated at 4° C in modified RIPA buffer containing 150 mM sodium chloride, 50 mM Tris Cl, pH 7.4, 1 mM EDTA, 1.0% NP-40, and 0.5% sodium deoxycholic acid the cells. Protein integrity is ensured using a cocktail of protease inhibitors with broad specificity for the inhibition of aspartic, cysteine, and serine proteases as well as aminopeptidases (0.1 mM AEBSF HCl, 0.08 µM Aprotinin, 5 µM Bestatin, 1.5 µM E-64, 2 µM Leupeptin Hemisulfate and 1 µM Pepstatin A). The following phosphatase inhibitors were also added: 1 mM NaF and 1 mM Na3VO4. Cell debris was removed by centrifugation and membrane filtration. Protein concentration was determined by Lowry assay using a commercially available kit. The protein concentration was adjusted to 1 mg/ml using RIPA buffer. Multi-purpose Whole Cell Lysates produced by Rockland Immunochemicals are derived from tissues and cell lines using highly refined extraction protocols to ensure exceptionally high quality, protein integrity and lot-to-lot reproducibility. All extracts are tested by SDS-PAGE using 4-20% gradient gels.