Figure 6.

Student results determination of steady state levels of Msh2 and the altered Msh2 protein. (A) Immunoblot analysis of Msh2 levels. Protein extracts containing hemagglutinin (HA) tagged versions of the wild type Msh2 protein and the Msh2-M707I protein along with a no epitope control extract were fractionated by SDS-PAGE (7% acrylamide separating gel) and transferred to a nitrocellulose membrane. Immunodetection was performed with mouse α-HA monoclonal primary antibody 12CA5 and α-mouse IgG HRP-conjugated secondary antibody. Images were formed on X-OMAT AR-5 Kodak film for 5 min. Lane 1, Msh2-M707I protein extracts; lane 2, wild-type Msh2p protein extracts; lane 3, no epitope protein extracts. The indicated band at 111 kD represents the Msh2-HA fusion proteins (Msh2p). Sizes were determined by comparison with prestained molecular weight standards (not shown). The identity of the high molecular weight (∼175 kD) cross-reacting band seen in all lanes was not determined. The lower molecular weight bands in lane 2 are likely to be Msh2p degradation products. (B) Coomassie staining of electrophoresed yeast protein extracts. Protein extracts were separated by SDS-PAGE as described above. A duplicate half of the gel was stained with Coomassie Blue R250 Stain, destained overnight, and dried under vacuum at 80ÿC for 1 h. The lanes are as follows: lane 1, NEB prestained protein markers; lane 2, Msh2-M707I protein extracts; lane 3, wild-type Msh2p protein extracts; lane 4, no epitope protein extracts; and lane 5, Invitrogen SeeBlue prestained standards. (Figure and legend courtesy of Ruth Tennen, Princeton University. Reproduced with permission.)