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

We have developed a method for accurate quantitative analysis and statistic al comparison of the relative contents of the dystrophin-glycoprotein compl ex (DGC) in skeletal muscle. This method was applied to compare DGC content s in slow (soleus) and in fast (extensor digitorum longus, EDL) rat skeleta l muscles. The quantitative analysis combines a modified bicinchoninic acid (BCA) assay with Western blotting and enhanced chemiluminescence (ECL). Th is combination allows the use of high levels of detergents and reducing rea gents essential for extracting DGC. In addition, the evaluation of the tota l amount of proteins in each sample makes it possible to have a reference a nd to accurately compare relative protein levels without using a specific s tandard. With a large gradient gel, we could concomitantly compare two grou ps (n = 9) and quantify all protein contents differing highly in their mole cular 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: dystroph in, beta-dystroglycan, and gamma-sarcoglycan levels were 130%, 110% and 120 % higher in the soleus, respectively. The differences observed in the expre ssion level of cytoskeletal associated protein (dystrophin) and transmembra nous anchorage components may correspond to a physiological response of the muscle fibers to duration, magnitude, and frequency of the imposed mechani cal loading.