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Cells Basic Copper Sulphate Extract Preparation for Intracellular Protein Immunodetection

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Basic Copper Sulphate(WSDTY) Cells were treated with 3 mM DFP and lysed in Triton X-100 lysis buffer containing 20 mM Tris-HCl pH7.4, 1% (v/v) Triton X-100, 150 mM NaCl, 1 mM EDTA, 2 μg/ml leupeptin, 2 μg/ml pepstatin, 10 μg/ml trypsin inhibitor, 44 μg/ml PMSF, 10 μM TLCK and complete mini EDTA-free protease inhibitor. After 10 min incubation on ice, the mixture was centrifuged at 1,000 g for 10 min at 4°C. The supernatant was then used for SDS-PAGE and Western blot detection of active caspase-3, HO-1 or Actin. After incubation under the conditions described above in experimental conditions section, the culture medium was removed, 10 times concentrated with centricon 3 kDa then secretion of pro-MMP1 or ADAMTS5 by chondrocytes was assessed by Western blot.

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asked Jun 3, 2019 in Preguntas Comerciales by anonymous

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