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Quantitative analysis of relative protein contents by Western blotting: Comparison of three members of the dystrophin‐glycoprotein complex in slow and fast rat skeletal muscle
Author(s) -
Chopard Angèle,
Pons Françoise,
Charpiot Philippe,
Marini JeanFrançois
Publication year - 2000
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(20000201)21:3<517::aid-elps517>3.0.co;2-9
Subject(s) - dystrophin , blot , glycoprotein , skeletal muscle , biology , microbiology and biotechnology , chemistry , biochemistry , gene , anatomy
We have developed a method for accurate quantitative analysis and statistical comparison of the relative contents of the dystrophin‐glycoprotein complex (DGC) in skeletal muscle. This method was applied to compare DGC contents in slow (soleus) and in fast (extensor digitorum longus, EDL) rat skeletal muscles. The quantitative analysis combines a modified bicinchoninic acid (BCA) assay with Western blotting and enhanced chemiluminescence (ECL). This combination allows the use of high levels of detergents and reducing reagents essential for extracting DGC. In addition, the evaluation of the total amount of proteins in each sample makes it possible to have a reference and to accurately compare relative protein levels without using a specific standard. With a large gradient gel, we could concomitantly compare two groups ( n = 9) and quantify all protein contents differing highly in their molecular masses (from 35 kDa to 427 kDa). Each experiment was triplicated and normalized; the two muscles were compared using the Mann‐Whitney test ( P < 0.001) to establish their protein content. The DGC relative levels for the slow muscle soleus and the fast muscle EDL differed significantly: dystrophin, β‐dystroglycan, and γ‐sarcoglycan levels were 130%, 110% and 120% higher in the soleus, respectively. The differences observed in the expression level of cytoskeletal associated protein (dystrophin) and transmembranous anchorage components may correspond to a physiological response of the muscle fibers to duration, magnitude, and frequency of the imposed mechanical loading.