Microtubule Binding InVitro
The SPNR protein is coprecipitated with murine testis microtubules and its associated proteins. Extracts prepared from as few as four mouse testes (approximately 400 ^g tissue) were incubated at 37°C in the presence of GTP and the microtubule-stabilizing drug taxol and then centrifuged at 100 000 X g to pellet microtubules. The pellets were washed, resuspended, and subjected to two further rounds of centrifugation and washing. A Coomassie-stained SDS-PAGE gel containing samples taken from the starting extract, each supernatant, and each pellet from a typical purification experiment is shown in Figure 5A. A great increase in tubulin concentration is seen in the first pellet, as well as an enrichment for high-molecular-weight MAPs. By the third pellet, very few of the proteins found in the starting extract were visible, except for tubulin and a few associated proteins. The enrichment for tubulin in this purification procedure was taxol dependent, as shown in Figure 5A. In the absence of taxol there was a slight enrichment of tubulin in the first pellet, but it was lost along with other proteins from the starting extract by the third round of purification. buy cipro
Western blots of SDS-PAGE gels identical to those stained with Coomassie were incubated with anti-SPNR and anti-kinesin antibodies (Fig. 5B). Both SPNR and kinesin were highly enriched in the microtubule pellets through three rounds of purification. This enrichment was also dependent on the presence of taxol, since there was a significant loss of each protein along with the loss of tubulin after further rounds of washing and centrifugation (Fig. 5B).
FIG. 5. SPNR coprecipitates with testis microtubules. A) Aliquots of the starting testis extract, each supernatant, and each pellet from a typical microtubule purification performed with (lanes 2-8) and without (lanes 10-16) taxol were run on SDS-PAGE gels and stained with Coomassie blue. B) Western blots of duplicate gels to those shown in A were probed with anti-kinesin and anti-SPNR antibodies. Molecular weight standards (X 10~3) are on the left (lanes 1 and 9).