198

1. Hours after release rec PR-Set7 Ladder kDa 0 15 0 15 198 115 93 49.8 PR-Set7 35.8 29.2 21.3 -PR-Set7 Coomassie Supplemental Figure 2. Specificity of the PR-Set7 antibody. Polyclonal anti-sera against PR-Set7 was raised by immunization of rabbits with recombinant PR-Set7. The IgG fraction was partially purified from sera and subjected to affinity purification using a column of recombinant C-PR-Set7 cross-linked to Affi-Gel 10 (Bio-Rad). Bound antibody was eluted with a pH-gradient and dialyzed against TBS. The specificity of the antibody was determined by Western analysis on HeLa whole cell lysate isolated from different times following release from G1 arrest; representing G1 (0 hours) and mitosis (15 hours). The left panel is the Coomassie stained gel with the approximate molecular weights indicated. The right panel is a Western of identical samples with the PR-Set7 antibody. The antibody detects two major bands: PR-Set7 (as indicated by the size of the recombinant PR-Set7 fusion protein) and a higher molecular weight band. The intensity of the higher molecular weight band does not change during cell cycle progression, whereas the PR-Set7 band was only detected during mitosis. This confirms that the antibody only detects PR-Set7 in the immunofluorescence studies (Figure 4C).