Subcellular Protein Fractionation and Immunoblotting Analysis
One-percent Triton X-100-soluble protein lysates and hypotonic protein extracts from freshly isolated spermatogenic cells were obtained as described. To separate cytosolic and membrane particulate fractions, the hypotonic extract from postnuclear supernatant was spun at 100 000 X g for 1 h at 4°C. The resulting supernatant is the cytosolic fraction while the pellet is the membrane fraction. The membrane fraction was solubilized by incubation in 2X SDS sample buffer for 15 min at 37°C and centrifuged; the supernatant was recovered. Protein concentrations were determined using Bio-Rad DC protein assay (Bio-Rad, Hercules, CA). Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Hybond-ECL, GE Healthcare) to be immunoprobed with the appropriate primary antibody. This was followed by treatment with the appropriate secondary, horseradish peroxidase-conjugated, immunoglobulins (IgGs) (GE Healthcare) and a chemiluminescence detection system (Pierce Chemical, Rockford, IL).buy actos online
Freshly prepared protein lysates, first preadsorbed to protein A-Sepharose beads (Sigma-Aldrich) and then clarified by centrifugation, were processed for immunoprecipitation assays. Clarified lysates (100 ig protein) were mixed with the appropriate antibody and incubated for 2 h, at 4°C, on a rotating platform, followed by the addition of protein A-Sepharose beads suspended in the lysate buffer, including a mixture of complete protease-inhibitor and phosphatase-inhibitor cocktails (Sigma-Aldrich) for a further 1 h. Preimmune serum was used for the control samples. After extensive washing (three times in 0.5% Triton X-100 in 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 1 mM ethylenediaminetetraacetic acid [EDTA], and once in 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 1 mM EDTA), the immunocomplexes were eluted by boiling in 2X SDS sample buffer to be then resolved by SDS-PAGE. After the blot transfer, the nitrocellulose membrane was incubated in the presence of the appropriate antibodies.