Fluorescent Labeling of Disulfide Proteins on 2D Gel for Screening Allergens: A Preliminary Study

An experimental protocol was established to detect disulfide proteins as fluorescent spots on 2D gel. In summary, free sulfhydryl groups in a protein mixture were capped with nonfluorescent iodoacetamide, followed by chemical reduction of disulfide bonds, and labeling of newly exposed sulfhydryl groups with the fluorescent probe, monobromobimane. Disulfide proteins were detectable as fluorescent spots under the UV lamp. As accumulating evidence suggests that disulfide bonds are responsible for the allergenicity of many proteins, we sought to use this protocol to find new allergens. In a model experiment using soybean trypsin inhibitor, a wellknown allergen with disulfide bonds, and myoglobin, which has a free sulfhydryl group, only the former was labeled with fluorescence. Application of the protocol to mite and pollen extracts facilitated detection of known allergens or putative allergens exhibiting sequence similarities to known allergens. In this note, we report the protocol as a complementary tool for screening allergens, which is now solely dependent on immunological recognition.