Seeking to find a highly sensitive antibody to detect human ubiquitin thats works well.
This mono-ab from Millipore work well: http://www.millipore.com/catalogue/item/mab1510
You would need to use 1:2000 or even 1:4000 if you work with WB on cancer cell lines (load 5ug of total protein).
Millipore also offers lys-48 and lys-63 specific antibodies.
what about ubi-1 clone ubiquitin Ab from Invitrogen (former Zymed)?or p4d1 from santa cruz ?did you try any of them?
I didn't try those. However p4d1 (Santa Cruz) are used at 1:50 (!!) dilution and my overall experience with dozens of other ab from Santa Cruz is very sad. That is the last place I ever look for antibody. I used some ab from Zymed and it worked good but that was not anti-ubq.
We use P4D1 from Santa Cruz, 1:1000 dilution in PBS and we never had a problem with detection. To detect specific linkages of chains (K48 and K63) we use the antibodies from cell signaling (also 1:1000) and they work also well.
Good to know that it worked for you, Selsen. Of course, because I didn't try p4d1 myself I only relied on publisher papers where people have used it at 1:50 to 1:500 dilution. I guess a lot depends what cells you were using and what was the loading.
Refat, for additional input regarding anti-ubq antobidies see this previous discussion on Research Gate: https://www.researchgate.net/post/Anybody_use_anti-ubiquitin_antibody_for_western_blotting_Suggest_antibody_to_detect_human_mouse_ubiquitin
I use the P4D1 from Santa Cruz. This antibody works well if you want to use an anti light chain Kappa antibody (if you have immunoprecipitation cross reactivity issues).
We have tested the P4D1 from Santa Cruz, and while it does recognise ubiquitin, it also reacts with the ubiqutin-like molecule Nedd8, in our hands. See attached pdf for figure.
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