2.3.1.48 | 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 |