A431 Whole Cell Lysate (EGF Stimulated)

Details for Product No. ABIN964022
Protein Species
Human
Species of Lysate
Human Cells
Application
Western Blotting (WB)
Options
Specificity The cells were grown in Dulbecco’s medium supplemented with 10% 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.
Characteristics Cell Line: Human A431 (epidermoid carcinoma)
Induction: EGF - Epidermal Growth Factor (50 ng/ml)
Lysate Fraction Whole Cell Lysate
Lysate Treatment EGF treated
Lysate Type Cell Lysate
Lysed Cells A431 Cells
Background Ready-to-use A431 Whole Cell Lysate EGF Stimulated 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.
Synonyms: Lysate, Cell Lysate, Stimulated Lysate, A431
Application Notes A431 Whole Cell Lysate - EGF Stimulated 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: EGF
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 A431 Whole Cell Lysate - EGF Stimulated 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
Supplier Images
SDS-PAGE (SDS) image for A431 Whole Cell Lysate (EGF Stimulated) (ABIN964022) Coommassie stained SDS-PAGE of 20 ?l of Human Derived A431 Whole Cell Lysate (Ready-t...
SDS-PAGE (SDS) image for A431 Whole Cell Lysate (EGF Stimulated) (ABIN964022) SDS-PAGE of A431 Whole Cell Lysate Stimulated with EGF (50ng/mL). Lane 1: reduced A43...