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Gel electrophoresis of giant proteins: Solubilization and silver‐staining of titin and nebulin from single muscle fiber segments
Author(s) -
Granzier Henk L. M.,
Wang Kuan
Publication year - 1993
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/elps.1150140110
Subject(s) - nebulin , titin , sarcomere , skeletal muscle , chemistry , gel electrophoresis , myofibril , biophysics , biochemistry , myocyte , anatomy , biology , microbiology and biotechnology
Abstract Giant proteins in the megadalton range (> 0.5 MDa) appear to play important structural and functional roles in striated muscle. Titin (∼ 3 MDa) is involved in the generation of resting tension and the assembly and stability of the sarcomere in skeletal and cardiac muscle tissues, while nebulin (∼ 0.7 MDa) is thought to regulate thin filament length in skeletal muscle. Sodium dodecyl sulfate (SDS)‐gel electrophoresis is an important tool in revealing the size, quantity and integrity of these giant proteins in muscle tissues. We report here a method for solubilizing, detecting and quantifying titin and nebulin from short segments of single fibers of the rabbit psoas muscle. Muscle proteins ranging from 15 kDa to 3MDa were resolved on 3.3–12% gradient polyacrylamide gels that were silver‐stained and quantitated by densitometry. Presoaking fiber segments in a low ionic strength pH 8.4 buffer enhances the amount of solubilized titin and nebulin. Solubilizing the presoaked fiber segments with SDS at 60°C for 60 s maximizes the amount of intact titin; solubilizing at higher temperatures causes extensive degradation of titin. Detection sensitivity is sufficient to study titin and nebulin in fiber segments as short as 120 μm.