Gel electrophoresis of giant proteins: Solubilization and silver‐staining of titin and nebulin from single muscle fiber segments

Henk L.M. Granzier, Kuan Wang

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90 引文 斯高帕斯(Scopus)

摘要

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.

原文英語
頁(從 - 到)56-64
頁數9
期刊Electrophoresis
14
發行號1
DOIs
出版狀態已發佈 - 1993
對外發佈

ASJC Scopus subject areas

  • 分析化學
  • 生物化學
  • 臨床生物化學

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