HeLa Cell Nuclear Extract (Doxorubicin Stimulated)
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- Protein Species
- Human
- Species of Lysate
- Human Cells
- Application
- Western Blotting (WB)
- Specificity
- The cells were grown in DMEM supplemented with 10% FBS (Fetal Bovine Serum). Cells were treated with 0.5 µg/ml Doxorubicin for 2 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.
- Characteristics
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Cell Line: HeLa - Human epidermoid carcinoma
Induction: Doxorubicin (0.5 µg/ml)
No test method can provide total assurance that the hepatitis B virus, hepatitis C virus, human immunodeficiency virus, or any other infectious agents are absent. Thus, all blood products, including purified proteins derived from human blood sources, should be handled at Biosafety Level 2 as recommended by the CDC\NIH manual entitled Biosafety in Microbiological and Biomedical Laboratories for potentially infectious human serum, blood specimens or proteins derived from same. Source material for the human blood product supplied to your facility has been tested for the detection of HIV antibody, Hepatitis B surface antigen, antibody to Hepatitis C, HIV 1 antigen(s), antibody to HTLV - I/II, and syphilis by FDA guidelines. All units were found to be non-reactive/negative for these tests. All human blood source material is collected in FDA licensed centers and is tested with FDA approved test kits. - Sterility
- Sterile filtered
- Lysed Cells
- HeLa Cells
- Lysate Type
- Cell Lysate
- Lysate Fraction
- Nuclear Extract
- Lysate Treatment
- Doxorubicin Stimulated
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VNN1 293T Cell Transient Overexpression Lysate(Denatured)VNN1 WB Whole Cell Lysate Overexpression Lysate HEK 293T Cells Reactivity: Human
CYP26A1 293T Cell Transient Overexpression Lysate(Denatured)CYP26A1 WB Whole Cell Lysate Overexpression Lysate HEK 293T Cells Reactivity: Human
Hep2 Whole Cell LysateWB Whole Cell Lysate Cell Lysate HepG2 Cells Reactivity: Human
NIH/3T3 Whole Cell Lysate (PDGF Stimulated)WB Whole Cell Lysate Cell Lysate 3T3 Cells PDGF Stimulated Reactivity: Mouse
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A431 Whole Cell Lysate (EGF Stimulated)WB Whole Cell Lysate Cell Lysate A431 Cells EGF treated Reactivity: Human
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- 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 µL depending on the size format of your gel.
- Comment
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Lysate Fractionation: Nuclear Extract
Lysate Stimulation: Doxorubicin
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)
- Handling Advice
- 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 µL depending on the size format of your gel.
- Storage
- -80 °C
- Expiry Date
- 3 months
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- Background
- Ready-to-use nuclear extracts 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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