A proteolytic method for evaluating O-GlcNAcylation on proteins of similar molecular weight to antibody heavy chain after immunoprecipitation.

Abstract Investigating a protein of interest that runs at the same molecular weight as antibody heavy chain is a frequent deterrent to its evaluation by immunoprecipitation. Methods of minimizing the detection of the immunoprecipitating antibody are available. However, these still present a barrier to evaluating if intracellular proteins are modified by the O-GlcNAc post-translation protein modification due to interfering glycosylation on antibodies. IdeZ protease specifically cleaves antibody at the hinge region, allowing collapse of the antibody fragments to 25 kDa after denaturation. Thus, this proteolytic method uniquely allows evaluation of O-GlcNAcylation of proteins of interest formerly obscured by antibody heavy chain.