HeLa Cell Nuclear Extract TNFa Stimulated

• Catalog Number: orb348688
• Category: Tools
• Description: HeLa Cell Nuclear Extract TNFa Stimulated
• Tested applications: WB
• Dilution range: WB: User Optimized
• Form/Appearance: Liquid (sterile filtered)
• Purity: The cells were grown in DMEM supplemented with 10% FBS (Fetal Bovine Serum). Cells were treated with 0.2 µg/ml TNFα for 30 min. 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.
• Conjugation: Unconjugated
• Hazard Information: Non-Toxic
• Source: Human
• Storage: Store HeLa Cell Nuclear Extract TNF alpha Stimulated at -70° C or COLDER. For extended storage, aliquot Nuclear Extract to minimize freeze/thaw cycles.
• Buffer/Preservatives: Preservative: None. Stabilizer: 10% (v/v) Glycerol. 1X SDS-PAGE Sample Buffer (62.5 mM Tris HCl, 2% SDS, 10% Glycerol and 0.005% bromophenol blue, pH 6.8)
• Alternative names: HeLa Cell Nuclear Extract TNFa Stimulated, HeLa Nu
• Note: For research use only
• Application notes: 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.
• Expiration Date: 12 months from date of receipt.