Heat-denatured protein analysis by SDS-PAGE with low concentration of SDS. in the extraction medium

Since SDS-PAGE is a powerful tool for separation of proteins according to the apparent molecular weight, the method has been used as a biological tool for more than 50 years. However, SDS-denatured proteins usually lose their native molecular structures and their biological activities. In addition, a 2% SDS can dissociate molecular complexes and aggregates of many proteins, information of the protein-protein interaction may be lost in the SDS-PAGE pattern. Therefore, we employed a low concentration (0% or 0.1%) SDS in the extraction medium for investigating heat-denatured processes of muscle proteins. Bovine muscle pieces were incubated at 40, 50, 60, 70 and 80°C for 30 sec, respectively, homogenized in the extraction medium, and centrifuged at 6,200 rpm for 5 min. The supernatants were subjected to SDS-PAGE. Myosin heavy chain (MHC), collagen, tropomyosin, and actin were aggregated sequentially during heat process, and the respective protein bands were lost in the SDS-PAGE pattern. For food allergy tests, dot blot analysis detected successfully fish egg proteins, however, SDS-PAGE and Western blotting using samples extracted with 0.05% SDS extraction medium failed to detect the fish egg proteins in the Western blot.