Preparation of Calpastatin Samples for Western Blotting

Determination of calpastatin levels in cells or tissues by Western blotting using antibodies, or by assay of calpain inhibitory activities in vitro, may be required to assess the role and the level of calpastatin, with respect to the levels of other components of the calpain system, in certain cell biological phenomena. Although calpastatin is resistant to heat or denaturants, it is extremely labile to cellular proteases (1 ). The wide range and heterogeneity of the apparent molecular masses of calpastatins (17–170 kDa), as revealed by Western blotting of extracts of different tissues and cells, is due both to degradation of the proteins during preparation, and also to alternative splicing and posttranslational modifications (2 –4 ). In addition to these factors, estimation of molecular weight is unusually difficult because calpastatins migrate abnormally slowly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), so that their molecular masses are overestimated by 40–60% (5 ). Full-length calpastatins (made up of domains L and 1–4) from most tissues of human, rabbit, pig, and others, migrate as 110–120 kDa proteins in SDS-PAGE. However calpastatin from bovine heart migrates as a protein of 140–170 kDa (1 ), probably because this calpastatin has an amino-terminal amino acid sequence (XL region) upstream of the previously assigned translation initiation codon (6 ). In contrast, erythrocyte calpastatins lack amino-terminal domains and migrate as 70-kDa proteins (7 ,8 ).