Fig. 4.

Stability and applications of affinity clamps. (A) Gel-filtration chromatograms of ePDZ-a before (dashed line) and after (solid line) heat treatment (2 h at 50°C). Gel filtration was performed by using a Superdex75 column (Amersham Biosciences) in PBS (pH 7.4). (B) The SPR sensorgrams of ePDZ-a before (dashed line) and after (solid line) the same heat treatment as in A. (C) Western blotting of wild-type ARVCF in mammalian cell lysates. Lysates of an MDCK cell line stably expressing human ARVCF (denoted as +) (41) and the parent cell line (−) were detected with ePDZ-b2 fused with alkaline phosphatase. mAb indicates a positive control with anti-ARVCF monoclonal antibody 4B1 (41). Left shows Coomassie brilliant blue staining, and Right shows Western blotting. (D) Pull-down (immunoprecipitation) of SUMO tagged with the ARVCF peptide from E. coli lysate by affinity clamps. The lysate was mixed with an affinity clamp immobilized to streptavidin magnetic beads. SDS/PAGE of the input (I), unbound (U), wash (W), and bound (B) fractions visualized with Coomassie brilliant blue staining are shown. “Beads” indicates a control experiment without an immobilized affinity clamp. “cpPDZFN” indicates a control experiment using cpPDZ fused to the unmodified FN3 scaffold. The position of the captured target is marked with the triangle for ePDZ-b2 (lane B), and the equivalent position is also marked for cpPDZFN. “Binder” indicates the position of ePDZ-b2 and cpPDZFN, and “SA” indicates the position of streptavidin.