Peculiarities of SDS-PAGE of Titin/Connectin

Abstract

Titin (also known as connectin) is a giant elastic protein of striated and smooth muscles of vertebrates. The molecular weight of its isoforms is 3.0–3.7 MDa in striated muscles and 0.5–2.0 MDa in smooth muscles. Titin was discovered 40 years ago using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). At the present time, this method has not lost its relevance but has undergone a number of modifications that improve visualization of giant titin isoforms in the gel. This chapter provides historical insights into the technical aspects of the electrophoresis methods used to identify titin and its isoforms. We focus on the peculiarities of the technique because of which titin molecules remain intact and its high molecular weight isoforms can be visualized. Electrophoretic testing of changes in titin content in muscles can be used in medical practice to diagnose pathological processes and evaluate effective approaches to their correction. was performed in vertical agarose- strengthened 2.1% polyacrylamide gel (8 × 10 × 0.1 cm). (1) m. soleus (control); (2) m. soleus (proteolysis, 1 h); (3) left ventricle of heart (control); (4) left ventricle of heart (proteolysis, 30 min). Proteolytic cleavage of titin was performed under the influence of endogenous muscular proteases. To this end, small pieces of muscle tissue (20–30 mg) were held for 30–60 min at 25–30°C. Then, 2–3 mg pieces were taken from the muscle sample and placed into solubilizing solution (10 mM Tris–HCl, 1.2% SDS, 10% glycerol, 2% β-mercaptoethanol or 75 mM DTT, 8–10 μg/ml of leupeptin or E64, pH 7.0) for the extraction and further electrophoretic testing of the proteins. T3300 is probably the proteolytic fragment of NT titin with molecular weight of ~3300 kDa.