Figure 2.—

Western blot analysis of target proteins bound to Abp1p SH3 domain affinity resin. Affinity chromatography using wild-type Abp1p SH3 domain (SH3 wild type), a mutant Abp1p SH3 domain with an N53A substitution (SH3 N53A), and a second mutant with a Y54A substitution (SH3 Y54A) as ligands. Increasing amounts of whole-cell extract (0.4, 0.8, and 1.6 mg) from wild-type log-phase cells expressing an HA-epitope-tagged version of either Abp1p SH3 target protein Ark1p (A) or Prk1p (B) were passed over the columns. Columns were washed; and bound proteins were eluted in buffer containing 1% SDS. Eluates were analyzed by Western blotting, using a monoclonal anti-HA antibody. (C) Affinity chromatography using Abp1p SH3 domain ligands described above and increasing amounts of purified GST-Srv2p-CTR protein (3, 6, and 12 μg). Columns were washed and bound proteins were eluted in buffer containing 1% SDS. Ten percent of the eluates were analyzed by Western blotting, using a monoclonal anti-GST antibody. The binding of GST-Srv2p-CTR to the SH3 Y54A column is the same as the binding of GST-Srv2p-CTR to resin alone (data not shown). Max., maximum.