I transfected cells with three different plasmids expressing p53 gene. I also had non-transfected cells. I performed western blot of the whole-cell lysates and probed for p53 using DO-1 antibody. In all 4 conditions, I detected a band at approx ~53 kDa which is expected as all samples contain wild-type p53. But also I detected a band of ~58 kDa seen only with the three transfected conditions?
Does anyone have an idea of what that band represents?
Many thanks in advance
Hi Sarah,
Phosphorylation will alter the mobility of p53, or it may be a splice variant.
Have you tried blotting against a p53-null cell line treated in the same manner? This will tell you if bands you are seeing are all p53.
Btw, in my personal experience, even monoclonal antibodies sold by reputable companies can have problems (I found that a well-established monoclonal against tubulin that was not the antibody I thought I was buying at all from CST). Be extra careful with your controls, and you will save yourself the trouble of chasing a red herring.
In my opinions, DO-1 antibody is very specific to p53 in WB, especially in p53 overexpressed cells. So I believe what you saw is a p53 band. If you have tag in your plasmids, the increased 5kDa is due to tag. Otherwise, it's due to posttranslational modifications such as ubiquitin or phosphorylation.
In my opinion overexpressing p53 in your cells may lead to the generation of an otherwise modified form beside endogenously expressed p53.
a) Do you see any induction of bands verified as p53 (control vs transfected sample)? Are possibly only new bands increased?
b) Do you know the complete sequence expressed in transfected cells (compared to the sequence of endogneously expressed p53) and are there possibly any differences (termination sequences, signal sequences, SNPs etc.?
- Badges
- Science topic