Figure 5. In vitro protein-protein interaction assay with GST-Phd isoform fusion proteins.

GST fusion proteins containing the complete coding sequence for either Phd, PhLP1, PhLOP1, PhLOP2, or a GST control were incubated with either Ni-NTA resin without bSUG1-6xHis (-SUG1) or with bSUG1-6xHis attached Ni-NTA resin (+SUG1). After washing four times, the bound proteins were eluted. The supernatants (S), washes (W1-W4) and eluate (E) were subjected to 10% SDS-PAGE, electrophoretic transfer to Immobilon-P, and immunoblot analysis with ECL detection on X-ray film. The numbers represent the standard molecular weight standards loaded with each SDS-PAGE.

(A) and (B) The GST-Phd, GST-PhLP1 and GST-PhLOP1 were detected with Phd monoclonal antibody (Mab 1D6). The GST-PhLOP2 and GST were detected with GST monoclonal antibody. (C) The membranes of Figure 5B were stripped and reprobed with 6xHis monoclonal antibody to verify and to detect the presence of the bSUG1-6xHis protein in each supernatant and eluant.