Spermatid Perinuclear Ribonucleic Acid: RESULTS(8)

13 Feb
2013

A Coomassie-stained SDS-PAGE gel containing aliquots of each supernatant and pellet is shown in Figure 7A. The vast majority of the added tubulin is pelleted under these conditions, and most of the starting material remains in the first supernatant. Figure 7B shows a corresponding Western blot probed with the a-His-tag antibody that recognizes the E. coli-produced SPNR. The SPNR protein present in the starting extract was pelleted with the MAP-free bovine brain microtubules through two rounds of purification. The pelleting of the His-tagged SPNR protein was entirely dependent on the presence of added tubulin, as shown in Figure 7B. As a control for the possible nonspecific “trapping” of SPNR in the microtubule pellet, the experiment described was repeated with and without the addition of NaCl to the starting extract. As shown in Figure 8A, the addition of NaCl at a concentration of 1 M did not interfere with the pelleting of tubulin through two rounds of purification.

However, as shown by Western blotting with the a-His-tag antibody (Fig. 8B), the addition of NaCl resulted in all of the SPNR protein remaining in the first supernatant, not in the tubulin-enriched pellet. Thus, it appears that the pelleting of His-tagged SPNR from E. coli extracts with MAP-free bovine brain microtubules is due to a specific salt-sensitive interaction with tubulin and is not due to a nonspecific trapping event. buy cheap antibiotics
Fig7Spermatid PerinuclearFIG. 7. SPNR binds to MAP-free bovine brain microtubules. A) MAP-free bovine brain tubulin was added to His-tagged SPNR-containing E. coli lysates. Microtubules were polymerized with the addition of taxol and GTP. Microtubules were pelleted, washed, and repelleted. Aliquots from each supernatant and pellet were run on SDS-PAGE gels and stained with Coomassie blue. A control experiment without the addition of tubulin to the E. coli extract was also performed. B) Western blots of duplicate gels to those shown in A were probed with an a-His antibody specific for the His-tagged SPNR protein. Molecular weight standards (X 10~3) are on the left.

Fig8Spermatid PerinuclearFIG. 8. A) MAP-free bovine brain tubulin was added to His-tagged SPNR containing E. coli lysates with and without NaCl added to a concentration of 1 M. Microtubules were polymerized with the addition of taxol and GTP. Microtubules were pelleted, washed, and repelleted. Aliquots from each supernatant and pellet 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 an a-His antibody specific for the His-tagged SPNR protein. Molecular weight standards (x 10~3) are on the left.

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