J. Anim Sci.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Doumit, M. E.
Right arrow Articles by Koohmaraie, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Doumit, M. E.
Right arrow Articles by Koohmaraie, M.

Journal of Animal Science, Vol 74, Issue 11 2679-2686, Copyright © 1996 by American Society of Animal Science

JOURNAL ARTICLE

Development of an enzyme-linked immunosorbent assay (ELISA) for quantification of skeletal muscle calpastatin

M. E. Doumit, S. M. Lonergan, J. R. Arbona, J. Killefer and M. Koohmaraie
Roman L. Hruska U.S. Meat Animal Research Center, ARS, USDA, Clay Center, NE 68933-0166, USA.

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 Immulon 4 (Dynatech) plates was observed when heated muscle extracts were diluted to 2 to 4 micrograms of protein/mL and incubated for 2 h at 37 degrees C. Optimum primary (30 micrograms IgG/mL) and secondary (Sigma A-6154; 1:1000 dilution) antibody incubations were for 1 h at 37 degrees C. Tetramethylbenzidine was used as substrate and A450 of the stopped reaction product was recorded in an automated plate reader. Calpastatin ELISA results were linearly related to calpastatin activity (calpain inhibitory activity) of heated longissimus muscle homogenates from prerigor lamb (r2 = .89; n = 40) and beef aged for 24 or 48 h (r2 = .90; n = 47). Intra-assay CV was < 5% (n = 8) and inter-assay CV was < 6% (n = 5). This assay offers advantages of speed, simplicity, and sensitivity over conventional methodology for calpastatin quantification.


This article has been cited by other articles:


Home page
 J DAIRY SCIHome page
K. A. Cummins, S. M. Lonergan, and E. Huff-Lonergan
Short Commuunication: Effect of Dietary Protein Depletion and Repletion on Skeletal Muscle Calpastatin During Early Lactation
J Dairy Sci, May 1, 2004; 87(5): 1428 - 1431.
[Abstract] [Full Text] [PDF]

Home page
 J ANIM SCIHome page
E. E. Helman, E. Huff-Lonergan, G. M. Davenport, and S. M. Lonergan
Effect of dietary protein on calpastatin in canine skeletal muscle
J Anim Sci, September 1, 2003; 81(9): 2199 - 2205.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 1996 by the American Society of Animal Science.