FIGURE 2.

Biochemical coupling of R1597W A2 to DNA handles. 802-bp DNA handles were PCR-amplified using 5′-thiol forward primers and 5′-biotin or -digoxigenin reverse primers. DNA handles were then activated using 2,2′-dithiodipyridine and reacted with freshly reduced R1597W A2 to disulfide link the handles to A2, as described previously (8). Disulfide coupling was verified by subjecting reaction products to 4–20% polyacrylamide Tris borate-EDTA gel electrophoresis with or without reduction with 1 mm DTT and staining with ethidium bromide. Shifts in migration upon coupling or binding to an antibody to C-terminal region of A2 (R&D Systems) report successful coupling of DNA handles to R1597W A2. A, DNA handles only; B, DNA handles + R1597W; C, same as B + A2 antibody. *, linkage to DNA was abolished under reducing conditions. DNA markers are shown on the left of each gel.