Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting comparisons of purified myofibrils and whole muscle preparations for evaluating titin and nebulin in postmortem bovine muscle.
Purified myofibril (MF) and homogenized whole muscle (WM) samples were prepared from A maturity market steers. Samples were removed at 0, 1, 3, 7, 14, and 28 d postmortem. The MF and WM samples from all steers were analyzed by SDS-PAGE (5% gels) and by Western blot analysis using monoclonal antibodies to titin and nebulin. The rates of degradation of the intact forms of titin and nebulin, with regard to differences dependent on sample type (MF vs WM), were examined. The results showed that there was very little difference in the rate of postmortem degradation of the intact form of titin or of intact nebulin with respect to the two types of samples examined. Analysis of MF and WM preparations revealed that titin and nebulin were progressively degraded, each at its own rate, with nebulin degrading faster, as postmortem storage time increased. Examination of MF and WM samples showed that the intact form of titin (T1) was absent at the same time postmortem in both sample types. Intact nebulin was not detected in MF and WM preparations at the same time postmortem with respect to sample type examined. Our results indicate that either purified MF or WM samples can be used satisfactorily to analyze the rate of degradation of the intact forms of both titin and nebulin.
Huff-Lonergan
E
E
Department of Animal Science, Iowa State University, Ames 50011, USA.
Mitsuhashi
T
T
Parrish
F C
FC
Robson
R M
RM
D000818:Animals; D015153:Blotting, Western / Q000379:methods / Q000662:veterinary; D002417:Cattle / Q000378:metabolism*; D064211:Connectin; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods / Q000662:veterinary; D008297:Male; D009124:Muscle Proteins / Q000032:analysis* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000378:metabolism; D009210:Myofibrils / Q000737:chemistry* / Q000378:metabolism; D011180:Postmortem Changes; D011494:Protein Kinases / Q000032:analysis* / Q000378:metabolism
D064211:Connectin; D009124:Muscle Proteins; C035620:nebulin; D011494:Protein Kinases