HeLa Cell Nuclear Extract (Etoposide Stimulated)
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- Espèce de la protéine
- Humain
- Espèce du lysat
- Human Cells
- Application
- Western Blotting (WB)
- Specificité
- 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.
- Attributs du produit
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Cell Line: HeLa - Human epidermoid carcinoma
Induction: Etoposide (10 µg/ml) - Stérilité
- Sterile filtered
- Lysed Cells
- HeLa Cells
- Lysate Type
- Cell Lysate
- Lysate Fraction
- Nuclear Extract
- Lysate Treatment
- Etoposide Stimulated
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- Indications d'application
- 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.
- Commentaires
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Lysate Fractionation: Nuclear Extract
Lysate Stimulation: Etoposide
Lysate Tissue Culture: Tissue Culture - Restrictions
- For Research Use only
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- 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)
- Conseil sur la manipulation
- 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.
- Stock
- -80 °C
- Date de péremption
- 3 months
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- Sujet
- 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.
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