FIGURE 2.

Differential capture of E. coli cell proteins on a wild-type ClpS affinity column. A, many E. coli proteins bind to wild-type ClpS and not to ClpS_DD/AA. Affinity resins were prepared with either wild-type ClpS or mutated ClpS in which Asp³⁵ and Asp³⁶ were changed to alanine. Extracts of cells harvested during stationary phase were clarified, and equal portions were loaded onto columns (1-ml bed volume) with either wild-type or mutated ClpS. The columns were washed with several column volumes of buffer, and proteins were eluted with buffer containing 1 mm FKTA-NH₂. Fractions of 0.5 ml were collected, and equal aliquots of four fractions that contained protein eluted from the wild-type column were mixed with SDS sample buffer and loaded onto an SDS-polyacrylamide gel (lanes labeled WT). Parallel fractions from the ClpS_DD/AA were loaded in adjacent lanes as indicated (lanes labeled AA). Proteins were detected by staining with Coomassie Blue. B, pulldown of proteins from E. coli cells carrying mutations in the N-end rule pathway. Clarified cell lysates from wild-type, ΔclpSA, or Δaat strains were loaded onto a ClpS affinity column, and bound proteins were eluted with FKTA. No proteins were detected in the FKTA eluate when wild-type lysates were applied to a control column with inactivated (Inact.) resin that had no cross-linked ClpS. Stds, standards.