Hep2 Whole Cell Lysate

• Cat.No.: Hep2-7H

Specification

• Species: Human
• Tag: Non
• Concentration: 2 mg/ml
• Tissue Type: HEp2 (HeLa contaminant)
• Preparation method: The cells were grown in MEM supplemented with 10% FBS (Fetal Bovine Serum). The lysate was prepared by first washing the cells in PBS supplemented with EDTA and a cocktail of protease inhibitors (see below). Washed cells are then incubated on ice in modified RIPA buffer containing 150 mM sodium chloride, 50 mM Tris Cl, pH 7.4, 1 mM EDTA, 1.0% NP-40, 0.25% sodium deoxycholic acid to lyse 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 (0.5 mM AEBSF HCl, 0.4 mM Aprotinin, 25 M Bestatin, 7.5 M E-64, 10 M Leupeptin Hemisulfate and 5 M Pepstatin A). Cell debris was removed by centrifugation. Protein concentration was determined by biuret assay using a commercially available kit. The protein concentration was adjusted to 2 mg/ml and then an equal volume of 2X SDS-PAGE sample buffer was added.
• Recommended Usage: For research use only, not for diagnostic or therapeutic use.
• Storage Buffer: 1X SDS-PAGE Sample Buffer (62.5 mM Tris HCl, 2% SDS, 10% Glycerol and 0.005% bromophenol blue, pH 6.8)
• Applications: Protein Lysate for WB. Ready-to-use lysates are especially prepared as positive controls for separation by SDS-PAGE and subsequent western blot analysis. Lysates are prepared in denaturing buffer WITHOUT dissociating agents (i.e. no 2- mercaptoethanol or dithiothreitol has been added).