Fig.2. Analysis of sequential steps in the purification of Gillichthys mirabilis Hsc70. (A) Silver-stained gel of various stages in the purification process. For silver staining, 5µg of total protein was applied to lanes 1–4 and separated by electrophoresis on a 7.5% SDS–polyacrylamide gel. Lanes are as follows: (1) silver-stained protein molecular mass standards; (2) 10700g supernatant from G. mirabilis white muscle; (3) pooled eluant from the DEAE anion-exchange column; (4) pooled eluant from the ATP affinity column (purified Hsc70 preparation); (5) 0.1µg of an Hsc70 standard purified from bovine brain. (B) Western blot detection of purified 70kDa heat-shock proteins using a rat monoclonal anti-Hsp70 antibody. Lane 1, 0.1µg of bovine brain Hsc70 standard; lane 2, 5µg of Hsc70 purified from G. mirabilis.