Fig 3.
Effect of DTT on GhCesA1 Zn domain dimerization. Size-exclusion HPLC of purified recombinant His-tagged GhCesA1 Zn domain (H-A1ZD) in the presence of Zn2+ (20 μM ZnCl2) under nonreduced (A) and (B) reduced conditions (5 mM DTT). The elution time (min) of the marker proteins lactalbumin (14.2 kDa) and carbonic anhydrase (29 kDa) are indicated. (C) H-A1ZD was preincubated with the indicated amounts of DTT for 10 min at 4°C and analyzed on 15% nondenaturing PAGE developed by silver staining. Monomer and dimer are indicated. (D) H-A1ZD, pretreated as described in C was incubated with monobromobimane, subjected to 15% SDS/PAGE, and fluorescent labeling was visualized at 365 nm.