Monday, November 24, 2003
Description Activation of apoptotic activity in cytoplasmic extracts for western blot analysis Procedure Activation reaction Cytoplasmic extracts were activated according to following protocol (as an example, cytochrome c/dATP are used as apoptotic stimuli). The components were mixed in the given order and then incubated at 37C usually for 1 h. x l Dilution Buffer (DB) containing ATP regeneration system + 0.46 l cytochrome c from bovine heart (325 M) + 0.3 l dATP (100 mM)+ y l cytoplasmic extract (75 g protein) x was so adjusted that the total volume was 30 l and thus the final protein concentration at 2.5 g/l. When active recombinant caspases (such as caspase-8) are used for activation of the extracts, 100 ng of recombinant caspase is added instead of cytoc/dATP. In case that the effect of inhibitors was studied, such as Ac-DEVD-fmk, or zVAD-fmk, the inhibitor at concentrations ranging from 100 nM up to 100 M was preincubated together with the extract in DB for 5 min, then cyto c/dATP was added. Western Blot Analysis After incubation of the extracts in the presence or absence of activating stimuli (cytochrome c/dATP or active recombinant caspase), western blots were performed loading usually 20 g protein in sample buffer per lane on a 4-20% SDS-PAGE gel Recipes Extract Dilution Buffer (DB): COMPOSITION: RECIPE for 500 l: 10 mM HEPES (pH 7.0) 5 mM EGTA 50 mM NaCl 2 mM MgCl2 1 mM DTT supplemented with ATP regeneration system: 2 mM ATP 10 mM phosphocreatine 50 g/ml creatine kinase 100 l of 50 mM HEPES, pH 7.0 incl. 25 mM EGTA 25 l of 1 M KCl 10 l of 100 mM MgCl2 0.5 l of 1 M DTT - 5 l of 200 mM ATP in water 10 l of 500 mM phosphocreatine in water 10 l of 2.5 mg/ml creatine kinase in KPM buffer 365 l H2O nuclease free Add DTT and ATP regeneration system always fresh to the buffer, just before use! -------------------------------------------------------------------------------- Caspase Assay Buffer (CAB): COMPOSITION: RECIPE for 50 ml: 50 mM PIPES 0.1 mM EDTA 10% glycerol 1 mM DTT 838.4 mg PIPES 10 l of 0.5 M EDTA 5 ml glycerol - add 40 ml H2O, adjust pH=7.2 using KOH (about 200 l of 1M KOH), then fill up to the final volume of 50 ml. 1mM DTT is always added fresh to the buffer, just before use. -------------------------------------------------------------------------------- Lysis Buffer (for DNA isolation from nuclei) COMPOSITION: RECIPE for 50 ml: 50 mM Tris-HCl, pH 8.0 10 mM EDTA 0.2 % SDS 5 ml of 0.5 M Tris-HCl, pH 8.2 1 ml of 0.5 M EDTA, pH 8.0 1 ml 10% SDS add 43 ml H2O before use, add proteinase K to a concentration of 0.5 mg/ml -------------------------------------------------------------------------------- TE Buffer COMPOSITION: RECIPE for 50 ml: 50 mM Tris-HCl, pH 8.0 1 mM EDTA 5 ml of 0.5 M Tris-HCl, pH 8.2 100 l of 0.5 M EDTA, pH 8.0 add 44.9 ml H2O -------------------------------------------------------------------------------- 4% Paraformaldehyde in PBS RECIPE for 100 ml: Heat 80 ml H2O to 60C under stirring add 4g Paraformaldehyde cover, let stir at 60C, but do not overheat add 2 drops of 1 N NaOH: solution becomes almost clear, but contains some particles that will not dissolve add 4 ml of 25x PBS adjust pH to 7.0 with HCl bring to final volume with H2O filter solution and store in brown glass bottle at 4C Solution is good for at least 1 year. Supplies Tips (责任编辑:泉水) |