Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, 66160, USA; Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, 66160, USA.
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, 66160, USA.
Anal Biochem. 2020 Dec 15;611:114001. doi: 10.1016/j.ab.2020.114001. Epub 2020 Oct 28.
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.
研究与抗体重链分子量相同的感兴趣的蛋白质，常常会对其免疫沉淀评估造成阻碍。有一些可以最小化检测到免疫沉淀抗体的方法。然而，由于抗体的糖基化干扰，这些方法仍然对评估细胞内蛋白质是否被 O-GlcNAc 翻译后蛋白修饰所修饰构成了障碍。IdeZ 蛋白酶特异性地在铰链区域切割抗体，使抗体片段在变性后collapse 为 25 kDa。因此，这种蛋白水解方法独特地允许以前被抗体重链掩盖的感兴趣的蛋白质的 O-GlcNAcylation 的评估。
