Identification and analysis of endogenous SUMO1 and SUMO2/3 targets in mammalian cells and tissues using monoclonal antibodies
Identification and analysis of endogenous SUMO1 and SUMO2/3 targets in mammalian cells and tissues using monoclonal antibodies
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DOI：
10.1038/nprot.2014.053
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发表时间：
2014-04-01
期刊：
影响因子：
14.8
通讯作者：
Melchior, Frauke
中科院分区：
文献类型：
--
作者：
Barysch, Sina V.;Dittner, Claudia;Melchior, Frauke
苏莫化是一种蛋白质修饰，调节数百种蛋白质的功能。检测内源性SUMO化很有挑战性：大多数小的泛素相关修饰物(SUMO)靶标丰度较低，而且蛋白质的细胞池中只有一小部分通常是SUMO化的。在这里，我们提出了一种从哺乳动物细胞和组织(特别是小鼠肝脏)中逐步浓缩内源性相扑靶标的方案，该方案基于科学界可用的单抗的使用。该方案包括(I)抗体和亲和基质的产生，(Ii)变性细胞裂解，以及(Iii)相扑免疫沉淀和多肽洗脱。亲和基质的产生和细胞裂解的过程类似于1d。免疫沉淀和多肽洗脱可以在2d内完成。由于SUMO蛋白是保守的，该方法也应该适用于其他生物，包括许多脊椎动物和黑腹果蝇。
SUMOylation is a protein modification that regulates the function of hundreds of proteins. Detecting endogenous SUMOylation is challenging: most small ubiquitin-related modifier (SUMO) targets are low in abundance, and only a fraction of a protein's cellular pool is typically SUMOylated. Here we present a step-by-step protocol for the enrichment of endogenous SUMO targets from mammalian cells and tissues (specifically, mouse liver), based on the use of monoclonal antibodies that are available to the scientific community. The protocol comprises (i) production of antibodies and affinity matrix, (ii) denaturing cell lysis, and (iii) SUMO immunoprecipitation followed by peptide elution. Production of affinity matrix and cell lysis requires similar to 1 d. The immunoprecipitation with peptide elution can be performed in 2 d. As SUMO proteins are conserved, this protocol should also be applicable to other organisms, including many vertebrates and Drosophila melanogaster.