Detection of giant myofibrillar proteins connectin and nebulin in fish meat by electrophoresis in 3-5% gradient sodium dodecyl sulfate polyacrylamide slab gels

An improved method was investigated for sodium dodecyl sulfate polyacrylamide slab gel electron phoresis (SIDS-PAGE) to facilitate the analysis of the giant myofibrillar proteins, connectin and nebulin, in fish meat by using jack mackerel (Trachurus japonicus) as the sample fish. It was established that separation of the alpha-connectin band from the beta-connectin band by SIDS-PAGE, could,be achieved by using 3-5% gradient gels with glycerol to facilitate the formation of a gradient with polymerization at 35 degreesC. SIDS-PAGIE samples of white dorsal muscle from the jack mackerel were homogenized with a 2% SIDS solution containing an inhibitor mixture (1 mug/mL of phenylmethanesulfonyl fluoride, 1 mug/mL of leupeptin, and 1 mug/mL of E-64) and heated at 50 degreesC for 20 min. Heating these samples at 100 degreesC for 2 min resulted in the disintegration of connectin but did not affect nebulin. A purified myofibril sample and a whole muscle sample showed similar changes in the overall rate of degradation of whole connectin and nebulin during the postmortem storage period, but it Was clear that beta-connectin was cleaved from alpha-connectin during the preparation of myofibrils at the early stage postmortem Storage of the SDS-PAGE samples at -85 degreesC was preferable to storage at -18 degreesC for a long period.