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CHO/K1 Whole Cell Lysate

WB Whole Cell Lysate Cell Lysate Reaktivität: Hamster
Produktnummer ABIN964019
  • Proteinspezies
    Hamster
    Lysatspezies
    Hamster
    Applikation
    Western Blotting (WB)
    Spezifität
    The cells were grown in Ham's F12 medium containing 2 mM L-Gln and 1.5 g/L sodium bicarbonate supplemented with 10% FBS (Fetal Bovine Serum). Cells were washed with PBS and then incubated on ice in modified RIPA buffer, containing 150 mM sodium chloride, 50 mM Tris HCl, pH 7.4, 1 mM EDTA, 1.0% NP-40, 0.5% sodium deoxycholic acid , 0.1% SDS and 0.01% (w/v) sodium azide to lyse the cells. Protein integrity was 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, 1 µM Pepstatin A). Phosphatase inhibitors 1 mM NaF and 1 mM Na3VO4 were also added. Cell debris was removed by centrifugation. Protein concentration was determined by a modified Lowry assay using a commercially available kit. Protein concentration was adjusted to 2 mg/ml and then an equal volume of 2X SDS-PAGE sample buffer was added.
    Produktmerkmale
    Cell Line: Chinese Hamster Ovary (CHO)
    Induction: None (Control)
    Lysate Type
    Cell Lysate
    Lysate Fraction
    Whole Cell Lysate
  • Applikationshinweise
    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.
    Kommentare

    Lysate Fractionation: Whole Cell Lysate
    Lysate Stimulation: Not Stimulated
    Lysate Tissue Culture: Tissue Culture

    Beschränkungen
    Nur für Forschungszwecke einsetzbar
  • Format
    Liquid
    Buffer
    1X SDS-PAGE Sample Buffer (62.5 mM Tris HCl, 2% SDS, 10% Glycerol and 0.005% bromophenol blue, pH 6.8)
    Handhabung
    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.
    Lagerung
    -80 °C
    Haltbarkeit
    3 months
  • Hintergrund
    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.
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