For various lines of evidence, I believe I have a new SUMO E3 ligase. But I need to validate it. As a matter of preference I don't like assays which over-express proteins in non-native cell types and then simply look to see if over-expression leads to the result you want.
So I move to in vitro biochemistry:
I know RanBP2 autosumoylates in vitro. And autoubiquitination is a general characteristic of Ub E3's. Can it be expected that SUMO E3s should all autosumoylate themselves? So should I do the in vitro assay, with no target, and do + or - my E3, if it is a true E3, should I then see it be autosumoylated?
Also, I do know some putative targets... but I like to do things in an unbiased method (hence part of my dis-like for the cell cultures methods), and I would like to test my E3s activity against total protein... to see if SUMOylation is increased when I add recomb. E3. Is this possible? Does anyone have a good method for this? I am assuming it is not as stupid easy as taking the in vitro SUMO kit, with my E3 (and w/o) and as target, add in total protein lysate. Any thoughts there?
Thanks in advance.
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