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Maus Embryonic Fibroblast Whole Cell Lysate

WB Whole Cell Lysate Cell Lysate Fibroblast Cells Reaktivität: Maus
Produktnummer ABIN964051
  • Proteinspezies
    Maus
    Lysatspezies
    Maus
    Applikation
    Western Blotting (WB)
    Spezifität
    The cells were grown in RPMI supplemented with 10% FBS (Fetal Bovine Serum). The lysate was prepared by first washing the cells in PBS. 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 (0.1 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 include 1 mM Sodium Fluoride and 1mM Na3VO4. Cell debris was removed by membrane filtration. 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.
    Produktmerkmale
    Cell Line: Mouse Embryonic Fibroblast
    Induction: None (Control)
    Sterilität
    Sterile filtered
    Lysed Cells
    Fibroblast Cells
    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
    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 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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