    2.3.1.32 | cells are lysed after centrifugation in 100 mM Tris-HCl buffer, pH 7.6, immunoprecipitation, affinity purification with ProFound c-Myc-Tag IP/Co-IP kit, subcellular fractionation of homogenized cells in 10 mM triethanolamine, 10 mM acetic acid, 250 mM sucrose, 1 mM EDTA, and 1 mM dithiothreitol, pH 7.4, centrifuged, membrane pellet resuspended in 5% Nycodenz and layered on top of a Nycodenz solution gradient in 10 mM HEPES, pH 7.4, fractions collected and further concentrated by ultracentrifugation |
| 2.3.1.32 | cells are washed with PBS, harvested in Tris-HCl, pH 7.4, containing 0.5% deoxycholic acid, 150 mM NaCl, 0.1% SDS, 4 mM EDTA, and 1% NP-40, with a protease inhibitor cocktail, 1 mM PMSF, 1 mM sodium fluoride, and 1 mM sodium orthovanadate, centrifugation, supernatant subjected to SDS-PAGE, or for immunoprecipitation lysed cells are collected with 50 mM Tris-HCl, pH 8.0, with 10% glycerol, 100 mM NaCl, 1 mM EDTA, and 0.1% NP-40 with proteinase inhibitor cocktail, 1 mM PMSF, 1 mM sodium fluoride, and 1 mM sodium orthovanadate, incubation with antibodies and G beads, washing, elution of proteins by boiling in 2X Laemmli loading buffer |
