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A431 Cell Nuclear Extract

WB Nuclear Extract Cell Lysate A431 Cells Reaktivität: Human
Produktnummer ABIN964034
  • Proteinspezies
    Human
    Lysatspezies
    Human Cells
    Applikation
    Western Blotting (WB)
    Spezifität
    The cells were grown in DMEM supplemented with 10% FBS (Fetal Bovine Serum). The lysate was prepared by first washing the cells in PBS. Washed cells were then incubated on ice in lysis buffer containing 10 mM HEPES, 60 mM KCl, 1.0 mM EDTA, 0.075% (v/v) NP40 and 1.0 mM DTT, pH 7.6. 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). Nuclei were then collected and washed in lysis buffer minus detergent. Nuclei were lysed by vortexing in extraction buffer containing 20 mM Tris-Cl, 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, and 25% (v/v) glycerol, pH 8.0, supplemented with protease inhibitors (see above). The lysate was clarified by centrifugation. Protein concentration was determined by Lowry assay using a commercially available kit. The protein concentration was adjusted to 2.0 mg/ml and then an equal volume of 2X SDS-PAGE sample buffer was added.
    Produktmerkmale
    Cell Line: Human A431 (epidermoid carcinoma)
    Induction: None (Control)
    Sterilität
    Sterile filtered
    Lysed Cells
    A431 Cells
    Lysate Type
    Cell Lysate
    Lysate Fraction
    Nuclear Extract
  • Applikationshinweise
    Ready-to-use nuclear extracts are especially prepared as positive controls for separation by SDS-PAGE and subsequent western blot analysis. Nuclear extracts are supplied in denaturing buffer without dissociating agents. Heat nuclear extract 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 10-30 l depending on the size format of your gel.
    Kommentare

    Lysate Fractionation: Nuclear Extract
    Lysate Stimulation: Not Stimulated
    Lysate Tissue Culture: Tissue Culture

    Beschränkungen
    Nur für Forschungszwecke einsetzbar
  • Format
    Liquid
    Konzentration
    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)
    Handhabung
    Ready-to-use nuclear extracts are especially prepared as positive controls for separation by SDS-PAGE and subsequent western blot analysis. Nuclear extracts are supplied in denaturing buffer without dissociating agents. Heat nuclear extract 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 10-30 µl depending on the size format of your gel.
    Lagerung
    -80 °C
    Haltbarkeit
    3 months
  • Hintergrund
    Ready-to-use nuclear extracts 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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