HeLa Whole Cell Lysate (Doxorubicin Stimulated)

Details for Product No. ABIN964036, Supplier: Log in to see
Protein Species
Human
Species of Lysate
Human Cells
Application
Western Blotting (WB)
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Specificity The cells were grown in DMEM supplemented with 10% FBS (Fetal Bovine Serum). Cells were treated with 0.5 µg/ml Doxorubicin for 2 h. Cells were washed in PBS and 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, 0.5% sodium deoxycholic acid and 0.1% SDS 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 (0.1 mM AEBSF HCl, 0.08 µM Aprotinin, 5 µM Bestatin, 1.5 µM E-64, 2 µM Leupeptin Hemisulfate and 1 µM Pepstatin A). Phosphatase inhibitors sodium fluoride, sodium orthovanadate, sodium pyrophosphate and -glycerophosphate were also added. Cell debris was removed by centrifugation. Protein concentration was determined by Lowry 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.
Characteristics Cell Line: HeLa - Human epidermoid carcinoma
Induction: Doxorubicin (0.5 µg/ml)
Sterility Sterile filtered
Lysate Fraction Whole Cell Lysate
Lysate Treatment Doxorubicin Stimulated
Lysate Type Cell Lysate
Lysed Cells HeLa Cells
Background Ready-to-use whole cell lysates produced are derived from cell lines or tissues 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 and immunoblot analysis using antibodies to key cell signaling components to confirm the presence of both high molecular weight and low molecular weight proteins.
Application Notes 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). Heat lysate to 95° C for 5 minutes and rapidly cool.  If dissociating conditions are desired, add reducing agent prior to heating.  The recommended loading volume per lane is 10-20 µl depending on the size format of your gel.
Comment

Lysate Fractionation: Whole Cell Lysate
Lysate Stimulation: Doxorubicin
Lysate Tissue Culture: Tissue Culture

Restrictions For Research Use only
Format Liquid
Concentration 1.0 mg/mL
Buffer 1X SDS-PAGE Sample Buffer (62.5 mM Tris HCl, 2% SDS, 10% Glycerol and 0.005% bromophenol blue, pH 6.8)
Handling Advice 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). Heat lysate to 95 °C for 5 minutes and rapidly cool. If dissociating conditions are desired, add reducing agent prior to heating. The recommended loading volume per lane is 10-20 µl depending on the size format of your gel.
Storage -80 °C
Expiry Date 3 months
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