Immunoblotting for Calpastatin Expression

Monica Averna; Roberta De Tullio

doi:10.1007/978-1-4939-8988-1_15

Immunoblotting for Calpastatin Expression

Methods Mol Biol. 2019:1915:195-206. doi: 10.1007/978-1-4939-8988-1_15.

Authors

Monica Averna¹, Roberta De Tullio^{2

3}

Affiliations

¹ Department of Experimental Medicine (DIMES)-Biochemistry Section, University of Genova, Genova, Italy. monica.averna@unige.it.
² Department of Experimental Medicine (DIMES)-Biochemistry Section, University of Genova, Genova, Italy.
³ Centre of Excellence for Biomedical Research (CEBR), University of Genova, Genova, Italy.

PMID: 30617805
DOI: 10.1007/978-1-4939-8988-1_15

Abstract

Immunoblotting is a procedure routinely used to analyze calpastatin expression. However, immunoblotting alone may not be adequate for this task, since calpastatin isoforms can vary by tissue, can be modified by partial digestion, and can undergo posttranslational modifications. Here we describe a method for more precise evaluation of calpastatin expression by combining immunoblot analysis with an assay for the inhibitory activity of a single calpastatin species isolated by SDS-PAGE and protein elution from the gel.

Keywords: Ca2+-dependent proteolysis; Calpain; Calpastatin; Calpastatin phosphorylation; Immunoblot; Protein elution.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Calcium-Binding Proteins / chemistry
Calcium-Binding Proteins / genetics*
Calpain / chemistry
Electrophoresis, Polyacrylamide Gel / methods*
Gene Expression / genetics*
Immunoblotting / methods*
Muscles / chemistry

Substances

Calcium-Binding Proteins
calpastatin
Calpain