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Isolation of Endogenous Calpastatin
Roberta De Tullio1,2, Monica Averna3
1Department of Experimental Medicine (DIMES)-Biochemistry Section, University of Genova, Genova, Italy. detullio@unige.it.
Methods in Molecular Biology (Clifton, N.J.)
|January 9, 2019
Summary
This study details the purification of calpastatin, a protein inhibitor, from human erythrocytes. The methods also yield human erythrocyte calpain and globin for assaying calpastatin inhibition activity.
Area of Science:
- Biochemistry
- Proteomics
- Human Physiology
Background:
- Calpastatin is a key inhibitor of calpain proteases.
- Assaying calpastatin activity requires purified calpain and Ca2+.
- Previous purification methods for calpastatin were often linked to calpain purification.
Purpose of the Study:
- To describe a method for purifying calpastatin from human erythrocytes.
- To provide a parallel purification protocol for human erythrocyte calpain.
- To enable reliable assaying of calpastatin inhibition activity.
Main Methods:
- Purification of calpastatin from human erythrocytes.
- Simultaneous purification of human erythrocyte calpain.
- Globin purification from human erythrocytes.
- Inhibition assays using purified calpastatin and calpain.
Main Results:
- Successfully purified calpastatin from human erythrocytes.
- Developed a method for co-purifying calpain and globin.
- Established a reliable system for measuring calpastatin inhibitory activity.
Conclusions:
- The described method provides purified calpastatin and essential components for its activity assay.
- This purification strategy facilitates the study of calpastatin-calpain interactions in human erythrocytes.
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