I am running a series of characterization experiments in different cell lines. When using the p53 [PAb 240] antibody from abcam (ab26) I obtain two bands around 50KDa. Surprisingly some lines have increased signal in the upper band and others in the lower (but all of them have the two bands), so I am intrigued. There are a lot of p53 modifications, is there anybody who can help me what to look for??
Splice variants of p53 (that's my prime suspicion: If you could show bands it would help to infer what it could be)
See this site
http://p53.free.fr/p53_info/p53_isoforms.html
OR monoubiquitinations
OR Altered confirmations due to phosphorylation (if it is minor variations)
OR K63/K48 Ubiquitinations (if you see a streak towards hight MW at higher exposures)
OR Antibody cross reactions (which is common for certain companies)
Good luck..!!
I agree with the previous response. Further, I would like to add that Pab240 antibody against p53 has been around for a long-time. Therefore, easiest thing to do would be to treat human normal cells with a DNA-damaging agent and see whether the intensity ratio of the two protein bands changes. If not, other possibilities need to be considered, including an alternative splicing variant. Notably, the antibody recognizes both mutant forms and wild-type human p53 under denaturing conditions.
What do you block with? I have found with cell lines that BSA can give an extra band there as well as 150kDa.
To block the membrane, we have been using 5% non fat dry milk (NFDM) prepared in TBST (Tween-20 final concentration 0.05%). The milk needs to be dissolved at 37 degree C for at least an hour before use. This blocking works for all antibodies (~700), which we have used in the last 20-years.
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