Casein zymography allows for specific, semiquantitative estimation of the μ-calpain activity.

Dilutions of commercial rabbit red blood cell μ-calpain (25, 50, 125, 250, 500 ng per well) were processed by casein zymography with or without preincubation with either calpeptin or calpastatin or calpain inhibitor II at concentrations indicated in “Patients, materials, and methods.” (A) Representative casein zymogram showing reduction of the band intensity proportional to reduced concentration of the enzyme. (B) A “dose-effect” curve relating concentration of the enzyme (x-axis, amount per lane) to the intensity of the casein-cleavage bands measured densitometrically (y-axis, arbitrary units); the relation allows for reliable estimation of relative activity of calpain in the range of 25 to 250 ng per lane. The curve summarizes 3 experiments and shows mean densities (AU) ± SD. (C,D) Inhibitory effect of calpeptin, calpastatin, and calpain inhibitor II on the activity of μ-calpain (50 to 250 ng per lane) estimated by casein zymography: a representative zymogram (C) and a bar graph (D) summarizing 3 experiments for the μ-calpain concentration of 50 ng per lane; bars represent arithmetic means (D).