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Ask help for ub assay in vivo
Posted by: popogirl (IP Hidden, New member, 9)
Date: October 16, 2005 05:36PM
Hi, everyone:
I am doing in vivo ubiquitination assay. I transfect cells with the plasmids expressing my interested protein and HA-ub. When I pull down my interested protein and probe with the specific Ab to my protein, I can easily detect my protein is ubquitinated( very clear smears show up). However, when I pull down HA-ub and then probe the Ab to my protein, I can any got single band rather than smears and the size is aroud the same as my protein. So I am wondering the latter reslut is consistent with the former one or contradict to it? why I can not get smears by latter method. Is any technique problem in my experiment? I am looking forward to any suggestion or help! Thank you very much!
Re: Ask help for ub assay in vivo
Posted by: EANON (IP Hidden, Junior member, 19)
Date: October 19, 2005 12:29AM
A definitive experiment is to IP your protein and probe for HA-Ub. I haven't heard of anyone IPing Ha-Ub and then probing for their protein.
In your first experiment, did you see smears or a clear ladder? Are you also transfecting the suspected E3 ligase?
Re: Ask help for ub assay in vivo
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: October 20, 2005 08:40AM
If you can prove the interaction in two-way direction it is more convincing. But often you will find it's difficult because there are some technical problems associated with the antibody quality and specificity, experiment conditions etc. So you will have to use other techniques to prove the interactions.
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