FIGURE 2.

SDS-PAGE analysis of gp5 under reduced and non-reduced conditions. The reducing agent (DTT) is removed from the protein sample using a G-25 column (see “Experimental Procedures”). A, lanes 1–4, non-reduced protein samples were preincubated with 1 mm DTT followed by mixing with the loading buffer containing 5 mm DTT. Lanes 5–8, non-reduced protein samples were mixed with the loading buffer lacking DTT to create non-reduced conditions during gel electrophoresis. Lanes 1 and 5, wild-type gp5; lanes 2 and 6, gp5-C275S; lanes 3 and 7, gp5-C313S; lanes 4 and 8, gp5-C275S/C313S. Gel electrophoresis was carried out on 4 - 20% linear gradient SDS-polyacrylamide gel. B, shown is the effect of trx on the disulfide bond formation. Polymerase was mixed with 50-fold excess of trx. Samples were then prepared and analyzed as described for panel A for non-reduced samples.