Fig. 10. Gel filtration chromatography of Ha-CalpM. A calpain fraction from Q Sepharose ion exchange chromatography was separated on a Superdex-200 gel filtration column (see Materials and methods). 10 µl samples of odd-numbered fractions were separated on 10% SDS-polyacrylamide gels and either stained with silver (A) or blotted onto PVDF membrane and probed with Ha-CalpM antiserum (B). The antibody recognized two Ha-CalpM isoforms of 62 kDa and 68 kDa (B, arrows) that co-eluted from the column. The smaller molecular mass immunoreactive polypeptides are probably degradation products. The arrow in A indicates the position of the 62 kDa isoform in the silverstained gel. The elution position of 62 kDa and 68 kDa isoforms (maximum at fraction 21) corresponded to that of CDP III (estimated mass 59 kDa) (Mykles, 2000; Mykles and Skinner, 1986). The peak CDP IIa activity (125 kDa) elutes at fraction 15 and the peak CDP IIb activity (195 kDa) elutes at fraction 12 (Mykles, 2000). Positions of molecular mass standards (kDa) are indicated.