in the assay of substrate ubiquitination of ACS5, why need ip...

锅锅 郭
Jul 14, 2022
protocol Protocol: In vitro Auto- and Substrate-Ubiquitination Assays
in the assay of substrate ubiquitination of ACS5, why need ip to pull down ACS5 then to test with ub antibody? just use YFP antibody to test if the RFP-ACS5 have ubiquitin, is that ok?
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Hye Lin Park Author Answered Jul 24, 2022

Department of Botany and Plant Pathology and the Center of Plant Biology, Purdue University, West Lafayette

Thank you for your question. We transiently expressed the YFP-ACS5 in tobacco leaves, and we did the immunoprecipitation to get rid of other tobacco proteins. Without IP, you can not check YFP-ACS5 is phosphorylated by MBP-SINAT2 or not, and we used UB-antibody to confirm the ubiquitination of YFP-ACS5. GFP-antibody is already used for IP process, so we can not use GFP-antibody for check the ubiquitination.
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