I am looking at ubiquitination of protein caused by inflammation. When I do my experiments in HEK293 cells with overexpression of my tagged protein, I see typical ubiquitin smear for pulldown samples of my protein. When I try to see ubiquitination of endogenous protein in pull down lysate from mouse tissue, I don't see any typical Ub smear (I do see smear for my positive control sample). Neither I see any difference in Ub bands for treatment vs. control.
I am using mouse Ub Ab to probe for ubiquitination. For HEK293 IP I am using tag specific Ab for pulldown. For endogenous protein pull down I am using polyclonal Ab against my protein.
Does anyone have experience on this? Do I need to use any DUB inhibitors or proteasome inhibitors for endogenous protein Ub detection? Why in most of the ubiquitination studies, people use HEK293 cells instead of other type of cell lines ?
First, are you sure that the smears correspond to ubiquitinated forms of your protein. You should perform your IP under denaturing conditions (boil your lysates in 1% SDS) to get ride off interacting partners. You should also try to IP using a a total Ub Ab (P4D1) and probe for your target protein. Second, over expressing conditions are artificial systems and can sometime be misleading. Finally, you can add DUB inhibitor (NEM) and protease inhibitor in your lysates in case.
First, are you sure that the smears correspond to ubiquitinated forms of your protein. You should perform your IP under denaturing conditions (boil your lysates in 1% SDS) to get ride off interacting partners. You should also try to IP using a a total Ub Ab (P4D1) and probe for your target protein. Second, over expressing conditions are artificial systems and can sometime be misleading. Finally, you can add DUB inhibitor (NEM) and protease inhibitor in your lysates in case.
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