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Development Of An Enzyme-Linked Immunosorbent Assay (Elisa) For Quantification Of Skeletal Muscle Calpastatin, M. E. Doumit, Steven M. Lonergan, J. R. Arbona, J. Killefer, M. Koohmaraie
Development Of An Enzyme-Linked Immunosorbent Assay (Elisa) For Quantification Of Skeletal Muscle Calpastatin, M. E. Doumit, Steven M. Lonergan, J. R. Arbona, J. Killefer, M. Koohmaraie
Steven M. Lonergan
An indirect antibody ELISA was developed for rapid and sensitive quantification of skeletal muscle calpastatin. Polyclonal antibodies were raised in rabbits against recombinant calpastatin, corresponding to domains 2, 3, and 4 of bovine skeletal muscle calpastatin. Western blot analysis revealed that these antibodies specifically recognize an immunoreactive calpastatin protein of approximately 130 kDa in prerigor skeletal muscle extracts. The intensity of the immunoreactive bands corresponds qualitatively with assayable calpastatin activity. For ELISA development, optimum dilutions of sample, primary anti-calpastatin antibody, and peroxidase-conjugated secondary antibody were determined by titration. A dilution optimum for coating of Immulonâ 4 (Dynatech) plates was observed …