Figure 1. Purification of recombinant βA3-crystallin by the nickel-affinity column chromatographic method. During purification of βA3-crystallin from the soluble protein fraction of the E. coli cell lysate, the bound proteins were eluted using 250 mM imidazole, and each fraction was analyzed using a 15% polyacrylamide gel using the SDS–PAGE method. Fractions (lanes 1 to 9) containing the single major protein band of βA3-crystallin were pooled, dialyzed, and used for further experiments. Circled bands 1, 2, and 3 were excised from gels, trypsin-digested and analyzed by quadruple ion trap (Q-TRAP) mass spectrometric method. The analysis identified these circled species (1, 2, and 3) as βA3-crystallin suggesting that the parent crystallin was partially degraded to produce two minor crystallin fragments during purification.