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Rat Testis Motor Proteins Associated with Spermatid Translocation: MATERIALS AND METHODS(3)

MATERIALS AND METHODS(3)

Controls for specificity of staining included replacing the primary antibody with normal mouse IgG used at the same concentration as the antibody, replacing primary antibody with buffer alone, and replacing both the primary and secondary antibodies with buffer alone. antibiotic levaquin

We also checked the IC74 and kinesin II antibodies for reactivity on immunoblots of isolated seminiferous epithelium. Epithelium, collected over a period of approximately 30 min as described elsewhere, was sedimented at low speed in a clinical centrifuge. The supernatant was discarded, and the pellet was resuspended in 250-500 ^l of PEM/ 250 (80 mM PIPES, 1.0 mM EGTA, and 1.0 mM MgCl2 [adjusted to pH 6.8 with KOH] and containing 250 mM sucrose) with and without protease inhibitors. To this was added 500 ^l of treatment buffer (0.125 M Tris-Cl pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol).

Samples were sonicated for 2 sec at 65 kHz and then aliquoted and stored at —20°. Aliquots at a protein concentration of approximately 1 mg/ml were processed for SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane using standard techniques, blocked overnight in buffer (0.05 Triz-ma-base adjusted to pH 7.5 with HCl, 0.25 NaCl, 0.1% Tween 20) containing 10% skim milk and 4% BSA, and then reacted with the dynein antibody at a 1:1000 dilution of the 5 mg/ml stock. Controls included replacing the primary antibody with normal mouse IgG at the same concentration or with buffer alone.