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文献和实验and measure protein concentration (BioRad assay) use 1 - 5 mg protein per IP immunoprecipitate for 2 h to ON, 4 °C wash immunoprecipitations pelleting the beads each time: 2 x 1 ml CHIP lysis buffer 2 x 1 ml CHIP
as given below: Low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH8.0, 150 mM NaCl); High salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH8.0, 500 mM NaCl); LiCl buffer (0.25 M LiCl, 1% NP-40, 1% SDC
In vitro Sphingomyelinase Assay
Reagents: Lysis buffer 25 mM Tris-HCl, pH 7.4 5 mM EDTA 1 mM ATP 20 µg/ml CLAP 1 mM PMSF Buffer A 10 mM MgCl2 0.2 M Tris-HCl, pH 7.4 0.2 % Triton X-100 Buffer B 0.2 M sodium acetate, pH 5.0 0.2 % Triton X-100
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