Figure 3. SDS-PAGE and Western blot analysis of detergent-skinned papillary muscle fibers reconstituted with RcTnT, RcT1-RfsT2 or RfsT1-RcT2.
(a) 2% SDS solution was used to digest the reconstituted fibers, as described previously 27. Equal quantities of digested protein samples (10 µg per sample) were run on 10% SDS-PAGE to achieve molecular weight-based separation of various sarcomeric proteins. The gel was then stained with Coomassie brilliant blue (R-250, Bio-rad Laboratories, Hercules, CA) to visualize the protein content. (b) Western blot. TnT was transferred from 10% SDS-gel to a PVDF membrane, and TnT in each preparation was probed using an anti-TnT primary antibody and an anti-mouse secondary antibody. Densitometric analysis of the TnT band profiles from the Western blot was carried using the ImageJ software (National Institutes of Health). Lanes 1–5 in (panels A and B) represent the following: lane 1, untreated cardiac fibers; lane 2, fibers reconstituted with wild-type c-myc tagged RcTnT; lane 3, fibers reconstituted with RcT1-RfsT2 chimera; lane 4, fibers reconstituted with RfsT1-RcT2 chimera and; lane 5, purified recombinant proteins. Tm stands for tropomyosin.