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Inter-laboratory optimization of protein extraction, separation, and fluorescent detection of endogenous rice allergens
In rice, several allergens have been identified such as the non-specific lipid transfer protein-1, the -amylase/trypsin-inhibitors, the -globulin, the 33kDa glyoxalase I (Gly I), the 52-63kDa globulin, and the granule-bound starch synthetase. The goal of the present study was to define optimal rice extraction and detection methods that would allow a sensitive and reproducible measure of several classes of known rice allergens. In a three-laboratory ring-trial experiment, several protein extraction methods were first compared and analyzed by 1D multiplexed SDS-PAGE. In a second phase, an inter-laboratory validation of 2D-DIGE analysis was conducted in five independent laboratories, focusing on three rice allergens (52kDa globulin, 33kDa glyoxalase I, and 14-16kDa -amylase/trypsin inhibitor family members). The results of the present study indicate that a combination of 1D multiplexed SDS-PAGE and 2D-DIGE methods would be recommended to quantify the various rice allergens.