Detection and quantification of the giant protein titin by SDS-agarose gel electrophoresis
Titin, a giant sarcomeric protein, is involved in the generation of passive tension during muscle contraction, assembly and stability of the sarcomere in striated muscles. Titin gene produces numerous titin protein isoforms with different sizes ( 3–4 MDa) resulting from alternative splicing. To study titin and titin isoform changes under disease conditions, the method to detect and quantify titin protein isoforms is needed. The method reported here is a 1% vertical SDS-agarose gel electrophoresis system that can solubilize, detect and quantify various titin isoform sizes. Sodium dodecyl sulfate (SDS)-agarose gel electrophoresis is an important tool in revealing the size and quantity of giant proteins in the sarcomere. In this method article, heart tissues were dissolved in urea-thiourea-glycerol sample buffer. Muscle proteins were resolved on 1% SDS-agarose gels that were silver-stained subsequently. Titin isoform bands with different sizes were separated on the gel. At the end, we also validated the method for large protein detection. Our results indicated that this electrophoresis method is efficient to study the transitions in titin isoforms. This method provides efficient protein extraction with urea-thiourea-glycerol buffer from hard tissues such as striated muscles This method provides an efficient way to separate large proteins over 500 kDa Combining with silver staining, our method can detect large protein isoforms and quantify the separated protein bands.