FIG. 4.

CzcD retention by metal affinity chromatography. Chelating Sepharose fast-flow columns were prepared with Zn²⁺ (A) or Mg²⁺ (as a negative control) (B). Sixty micrograms of CzcD protein was applied to each column, and the columns were washed with 10 bed volumes of 10 mM sodium phosphate buffer (pH 7.2). Fractions (300 μl) were collected and applied to an SDS—15% polyacrylamide gel, which was silver stained. Lanes 1 to 5 contained the protein from the first five wash fractions, and lanes 6 to 9 contained the first four elution fractions. The positions of size markers are indicated on the left.