Gelsolin Digestion of Testicular Fractions Enriched for Elongate Spermatids with Attached Sertoli Cell Junction Plaques
Testicular fractions enriched for spermatid/junction complexes were collected generally as described elsewhere. In brief, the protocol involved manually stripping epithelium from tubule walls and then mechanically fragmenting the epithelium by aspiration through a fine-bore pipette tip. The epithelial fragments were then loaded onto step sucrose gradients and centrifuged, and fractions enriched in spermatids with attached junction complexes were collected at one of the interfaces. antibiotics levaquin
For each of three experiments, and to ensure sufficient material for Western blot analysis, spermatid/junction complexes were collected and pooled from three animals. Three sucrose gradients were run for each animal, and material from the appropriate interfaces was pooled and stored on ice as wet pellets. When pellets had been obtained from all three animals, the pellets were resuspended in MES (2-(4 morpholino)-ethane-sulfonic acid) buffer (50 mM MeS-KOH pH 6.3, 2 mM MgCl2, 0.1 mM CaCl2 0.5 mM di-thiothreitol [DTT]) and pooled. The spermatid/junction complexes were washed once in MES buffer, centrifuged at 4000 rpm for 2 min in an Eppendorf bench-top centrifuge (Hamburg, Germany), and resuspended in MES buffer.