To assess binding to mouse testis microtubules, microtubule pellets from the first round of purification (described above) were resuspended in 120 ^l His-SPNR E. coli extract. The mixture was incubated at 37°C for 15 min, and microtubules were pelleted by spinning at 30 000 X g for 20 min at 37°C. The pellets were resuspended in MT buffer and subjected to another round of purification. Aliquots of the starting extract, the supernatants, and the microtubule pellets were analyzed by SDS-PAGE, followed by Coom-assie staining or Western analysis with anti-His-tag antibodies. buy ortho tri-cyclen
Two hundred micrograms of bovine brain tubulin that had been purified by phosphocellulose chromatography (kindly provided by Joe Howard, University of Washington, Seattle) was added to 100 ^l His-SPNR E. coli extract. Taxol was added to a final concentration of 20 ^m, GTP was added to a concentration of 1 mM, and the mixture was incubated at 37°C for 15 min. The microtubules were pelleted as described above. The pellets were resuspended in MT buffer and subjected to another round of purification. Aliquots from each supernatant and pellet were run on SDS-PAGE gels, followed by Coomassie staining or West-em analysis with anti-His-tag antibodies. Controls included the omission of tubulin and the addition of 1 M NaCl to the starting extract.