In my lab we are trying to detect ubiquitination on a membrane protein in yeast. We can see bands corresponding to the size of the mono-, di- and tri-ubiquitinylated form of the protein on Western blot when we use an antibody against the protein. However, we haven't been able to get a commercial anti-Ub-antibody which gives nice bands. (Tried one recommended for yeast by Santa Cruz.)
Do you have any suggestions or recommendations for antibodies against Ub that work well in yeast?
Here there are a revision of antibodies (not in yeast)
http://www.sciencedirect.com/science/article/pii/S0022283612003981
but we triedthe p4D1 from santacruz and is fine.
sorry is this Paper
http://www.ncbi.nlm.nih.gov/pubmed/22004789
As we know well, our experiments are as good as the antibody that we use... Try VU-1 mouse monoclonal ubiquitin Ab from LifeSensors that recognizes mono- and polyubiquitylated proteins as well as free ubiquitin, an important marker of cellular homeostasis. VU-1 is 'one reagent-multiple application' tool: you can use it for IHC/IF in tissue and cell culture as well as for Western blot. Since ubiquitin is very conserved between species, VU-1 works well in yeast. Interested in purification of polyubiquitylated proteins bud didn’t get results with IP using Ubi antibody? Try TUBEs (LifeSensors) for the pulldown.
http://www.lifesensors.com/pdf/VU101_VU-1_MAb_DataSheet_062212.pdf
Hi Maria, I have the same problems than you using p4d1 antibody. We try to analyse Rpb1 ubiquitinilation but this antibody does not works. We are not able to detect any band and the westerns are not nice, in contrast to other antibodies.
Have you found any other good antibody?
Victoria you mentioned the Lifesensor Tubes. Have you used them in yeast and if so did you also purchase the PR-619 ( protease inhibitor) and the 1, 10-phenanthroline ( metal chelator) from them?
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