Polyclonal anti α tubulin antibody

Aliquots of 5 × 10⁵ spermatozoa from wild-type and transgenic mice were incubated for various time points at 37°C in a humidified incubator with 5% CO₂ in noncapacitating and capacitating media. After incubation, sperm was centrifuged at 5 min at 10,000 rpm, and resuspended in lysis buffer (1 mM orthovanadate, 1 mM EDTA, 1 mM NaF, 1% NP-40, 1% deoxicolate, 0.1% SDS, and 1 × Phosphostop (Roche, Indianapolis, IN)). Samples were subjected to SDS-PAGE (7.5% gel) and immunoblotted, as previously described (Wagoner et al. 2005 (link)). Briefly, the membrane was blocked using 5% BSA in Tris-buffered saline with 0.5% Tween-20 (TBS-T) for 1 hour. Phosphorylation was detected by probing overnight at 4°C with anti-phosphotyrosine antibody (4G10 antibody; 1:1000 dilution in TBS-T Millipore, Billerica, MA. Blots were then wash 3 times with TBS-T, and then incubated for 1 hr with donkey anti-mouse IgG conjugated to horseradish peroxidase 1:1000 dilution in TBS-T. Following extensive washing, positive bands were detected using chemiluminescence. To ensure equal protein loading, membranes were stripped and re-probed with a polyclonal anti-α-tubulin antibody (1:1000 dilution) (Sigma-Aldrich). Phosphorylation intensities of the samples were analyzed with Pro-Gel Analyzer 4.5, and reported relative to the non-capacitated samples.