HCT-116 Cell Nuclear Extract

• Catalog Number: orb348713
• Category: Tools
• Description: HCT-116 Cell Nuclear Extract
• Tested applications: ChIP, IP, WB
• Concentration: 4.0 mg/ml
• Dilution range: ChIP: User Optimized, IP: User Optimized, WB: User Optimized
• Form/Appearance: Liquid (sterile filtered)
• Purity: HCT-116 cells were grown in McCoy'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 4 mg/ml in RIPA buffer containing protease and phosphatase inhibitors.
• Conjugation: Unconjugated
• Hazard Information: Non-Toxic
• Source: Human
• Storage: Store vial at -70° C or COLDER. For extended storage, aliquot Nuclear Extract to minimize freeze/thaw cycles.
• Buffer/Preservatives: Preservative: None. Stabilizer: None. 1X RIPA Buffer with HALT Protease and Phosphatase Inhibitors
• Alternative names: HCT-116 lysate nuclear extract, cell lysate, HCT11
• Note: For research use only
• Application notes: Multi-purpose HCT-116 nuclear extracts are especially prepared as positive control for multiple assays including western blot, immunoprecipitation (IP), capture ELISA or other assays requiring native protein sample. For separation by SDS-PAGE and subsequent western blot analysis, lysates should be diluted by user to desired concentration in SDS-PAGE buffer with 2-mercaptoethanol or dithiothreitol as the reducing agent and heated to 95° C for 5 minutes. Sample is ready for use in immunoprecipitation and ELISA experiments, conditions should be optimized by the user. Rockland recommends its Trueblot IP reagents for immunoprecipitation experiments.
• Expiration Date: 12 months from date of receipt.