Figure 1.

Presence of Mb polypeptide in Antarctic icefishes. (a) Soluble polypeptides were liberated from heart ventricles and resolved by SDS/PAGE. Lanes: 1, protein molecular weight standards (Bio-Rad); 2, human heart Mb standard (1 μg; Biodesign International, Kennebunkport, ME); 3, C. aceratus (35 μg total protein); 4, C. rastrospinosus (35 μg total protein). (b) Duplicate gel to a above was electroblotted to PVDF membrane for immunoblot analysis. The PVDF membrane was incubated with mouse anti-human Mb monoclonal antibody (Sigma). Bound antibody was detected by rabbit anti-mouse IgG antibody covalently linked to alkaline phosphatase (Bio-Rad) and subsequent incubation with stabilized Western blue substrate (Promega). Only the molecular weight range containing Mb is shown. Lane assignments are described as in a above. (c) Slot immunoblot of soluble polypeptides from heart ventricles of eight channichthyid icefishes (1A to 2C) and one red-blooded Antarctic nototheniid fish (2D). Equal amounts (5 μg) of total protein were loaded in each slot and incubated with antibody as described in b above. 1A, C. rastrospinosus; 1B, P. macropterus; 1C, P. georgianus; 1D, C. aceratus; 1E, C. hamatus; 2A, C. gunnari; 2B, C. wilsoni; 2C, C. antarcticus; 2D, N. coriiceps.