After the second centrifugation, the spermatozoa were diluted 1:10 in fresh TALP medium and sonicated as described above for the epididymal spermatozoa. ventolin 100 mcg
Antibodies against Scinderin
Rabbit polyclonal anti-scinderin #6 was raised against bovine scinderin and has been previously characterized.
Electrophoresis and Immunoblotting
Twenty-five micrograms of proteins of each tissue and cell homogenate were loaded in a 10% polyacrylamide minigel. Proteins were subjected to electrophoresis and electrotransferred onto nitrocellulose membranes as previously described. Membranes were blocked with 5% skim milk in TBS (140 mM NaCl, 50 mM Tris HCl, pH 7.4) and incubated with a scinderin antiserum #6 (1/300 dilution in 5% skim milk-TBS) for 2 h at 37°C. After extensive washing, membranes were incubated with alkaline phosphatase-conjugated anti-rabbit IgG (1/3000 dilution, in 5% skim milk in TBS) for 1 h at room temperature (RT). The reaction was developed by treatment with a mixture of p-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate toluidine salt.