HeLa Cell Nuclear Extract (Etoposide Stimulated)

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Proteinspezies
Human
Lysatspezies
Human Cells
Applikation
Western Blotting (WB)
Optionen
Hersteller
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Spezifität The cells were grown in Eagles medium supplemented with 10% FBS (Fetal Bovine Serum). Cells were treated with 10 µg/ml Etoposide for 13 h. 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: HeLa - Human epidermoid carcinoma
Induction: Etoposide (10 µg/ml)
Sterilität Sterile filtered
Lysate Fraction Nuclear Extract
Lysate Treatment Etoposide Stimulated
Lysate Type Cell Lysate
Lysed Cells HeLa Cells
Hintergrund Ready-to-use HeLa Cell Nuclear Extract Etoposide 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.
Applikations-hinweise 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 ul depending on the size format of your gel.
Kommentare

Lysate Fractionation: Nuclear Extract
Lysate Stimulation: Etoposide
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 ul depending on the size format of your gel.
Lagerung -80 °C
Haltbarkeit 3 months
Bilder des Herstellers
 image for HeLa Cell Nuclear Extract (Etoposide Stimulated) (ABIN964040) HeLa Cell Nuclear Extract (Etoposide Stimulated)
SDS-PAGE (SDS) image for HeLa Cell Nuclear Extract (Etoposide Stimulated) (ABIN964040) Coommassie stained SDS-PAGE of 25 ?g of Etoposide stimulated Human Derived Hela Cell ...