Gel electrophoresis of giant proteins: solubilization and silver-staining of titin and nebulin from single muscle fiber segments.
Giant proteins in the megadalton range (> 0.5 MDa) appear to play important structural and functional roles in striated muscle. Titin (approximately 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 (approximately 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 3 MDa 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 degrees 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 microns.
Granzier
H L
HL
Clayton Foundation Biochemical Institute, Department of Chemistry and Biochemistry, University of Texas, Austin 78712.
Wang
K
K
D000818:Animals; D002021:Buffers; D064211:Connectin; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009124:Muscle Proteins / Q000032:analysis* / Q000737:chemistry; D009132:Muscles / Q000737:chemistry*; D009994:Osmolar Concentration; D011494:Protein Kinases; D011817:Rabbits; D016622:Silver Staining / Q000379:methods*; D012995:Solubility; D013890:Thiourea; D014508:Urea
D002021:Buffers; D064211:Connectin; D009124:Muscle Proteins; C035620:nebulin; D014508:Urea; D011494:Protein Kinases; D013890:Thiourea