HeLa Cell Nuclear Extract Lysate
Référence OCRA00101
Conditionnement : 200ug
Marque : Aviva Systems Biology
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| Datasheets/Manuals | Printable datasheet for OCRA00101 |
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| Predicted Species Reactivity | Human |
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| Product Format | Liquid (sterile filtered) |
| Conjugation | Unconjugated |
| Application | Sodium sodecyl sulfate - polyacrylamide gel electrophoresis, Western blot |
| Additional Information | Cell Line: HeLa - Human epidermoid carcinoma |
| Reconstitution and Storage | Store HeLa Cell Nuclear Extract at -70°C or COLDER. For extended storage, aliquot Nuclear Extract to minimize freeze/thaw cycles. Expiration is three (3) months from date of opening. |
| Concentration | 1 mg/ml |
| Application Info | 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. |
| Preparation | 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 uM Bestatin, 1.5 uM E-64, 2 uM Leupeptin Hemisulfate, 1 uM 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. |
| Description of Target | Ready-to-use nuclear extracts produced by Rockland Immunochemicals 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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