Calpain pp 187-194 | Cite as

Isolation of Endogenous Calpastatin

  • Roberta De TullioEmail author
  • Monica Averna
Part of the Methods in Molecular Biology book series (MIMB, volume 1915)


We here describe the purification of calpastatin from human erythrocytes. When calpastatin is purified from tissues, it is necessary to measure its inhibitory activity against calpain in the presence of Ca2+ to specifically identify the protein. Thus, the purification steps necessary to obtain the inhibitor protein were originally designed to obtain calpain from the same tissue. For this reason, in addition to calpastatin purification, we also include a method for purifying human erythrocyte calpain and globin. We routinely use these two components for assaying calpastatin inhibition.

Key words

Calpastatin isolation Calpain inhibition Chromatography Calpain assay Human erythrocytes 



This work was supported by the grant FRA2015 and FRA2016 from the University of Genova to MA and RDT.


  1. 1.
    Pontremoli S, Viotti PL, Michetti M, Salamino F, Sparatore B, Melloni E (1992) Modulation of inhibitory efficiency of rat skeletal muscle calpastatin by phosphorylation. Biochem Biophys Res Commun 187:751–759CrossRefGoogle Scholar
  2. 2.
    Salamino F, Averna M, Tedesco I, De Tullio R, Melloni E, Pontremoli S (1997) Modulation of rat brain calpastatin efficiency by post-translational modifications. FEBS Lett 412:433–438CrossRefGoogle Scholar
  3. 3.
    Adachi Y, Ishida-Takahashi A, Takahashi C, Takano E, Murachi T, Hatanaka M (1991) Phosphorylation and subcellular distribution of calpastatin in human hematopoietic system cells. J Biol Chem 266:3968–3972PubMedGoogle Scholar
  4. 4.
    Barnoy S, Kosower NS (2007) Calpastatin in rat myoblasts: transient diminution and decreased phosphorylation depend on myogenin-directed myoblast differentiation. Int J Biochem Cell Biol 39:253–261CrossRefGoogle Scholar
  5. 5.
    Udenfriend S, Stein S, Böhlen P, Dairman W, Leimgruber W, Weigele M (1972) Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science 178:871–872CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Experimental Medicine (DIMES)—Biochemistry SectionUniversity of GenovaGenovaItaly
  2. 2.Centre of Excellence for Biomedical Research (CEBR)University of GenovaGenovaItaly

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