Hydrogen Peroxide Stimulated MCF-7 Whole Cell Lysate

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Hydrogen Peroxide Stimulated MCF-7 Whole Cell Lysate

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Cat.No. : MCF-7-18H
Species : Human
Concentration : 2 mg/ml
Tissue Type : Epithelial (Mammary)
Preparation method : The cells were grown in DMEM supplemented with 10% FBS (Fetal Bovine Serum). The lysate was prepared by first washing the cells in PBS. Washed cells are 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 and 0.1% SDS to lyse the cells. 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 M Aprotinin, 5 M Bestatin, 1.5 M E-64, 2 M Leupeptin Hemisulfate, 1 M 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 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.
Recommended Usage : For research use only, not for diagnostic or therapeutic use.
Storage Buffer : Lysate is supplied in SDS sample buffer containing 5% -mercaptoethanol
Applications : Protein Lysate for WB. Ready-to-use lysates are especially prepared as positive controls for separation by SDS-PAGE and subsequent western blot analysis. Lysates are prepared in denaturing buffer WITHOUT dissociating agents (i.e. no 2- mercaptoethanol or dithiothreitol has been added).

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