Figure 9
Electrophoresis gel showing expression of HA-tagged midasin in haploid yeast Preparations made from wild type yeast (wt) and from yeast with a HA epitope tag on the MDN1 gene (HA). Gel A: S0, supernatant fraction after homogenizing yeast with glass beads and centrifuging at 2000 × g for 2 min; S1, P1, supernatant and pellet fractions after centrifugation of S0 fraction at 18,000 × g for 20 min. Gel B: S2, P2, supernatant and pellet fractions obtained by resuspending P1 fraction in 8 M urea and centrifuging at 150,000 × g for 20 min. The midasin polypeptide (arrow) migrates somewhat more slowly than the dynein heavy chain (dyn) used as a molecular mass standard of ~525 kDa. The 45 kDa band in the 8 M urea supernatant is a cross-reacting protein present in wild type yeast. The 3–8% polyacrylamide gels were electrophoresed in the presence of 0.1% Na dodecyl SO4 and blotted onto Immobilon membrane. The blot was stained with monoclonal antibody against the HA epitope. The molecular mass standards were run in a noncontiguous lane of gel A.