I am not getting p53 expression in western blot. I loaded more protein 40 microgram and primary antibody dilution was 1:500 still didn't get expression. Where have I gone wrong?
what buffer did you use to extract protein? did you add inhibitor of proteases? which cells did you use? do they express WT or mutant p53? which primary p53 antibody did yo use? is it a mouse, sheep, rabbit antibody? did you use the appropriate secondary antibody? did you check that the trasnfer went well by staining the nitrocellulose membrane with red ponceau? can you detect any other protein (i.e. actin) by western-blot?
Hi Devashree, which p53 antibody did you use at 500x dilution. I have checked p53 expression in many different cell types including primary samples. Assuming that there is no problem is your sample preparation and transfer, I would recommend you to use Mouse monoclonal anti-p53 antibody (DO-7) which can detect both wild type and mutant p53 antibody. You can get it from any companies, however, from my experience this parituclar antibody from Sigma works much better in terms of quality and specificity. This antibody gets strong single band for p53 even at 1:10,000 dilution. I hope it will help. Thanks.
I am using Mouse monoclonal anti p53 (DO-1) of Santacruz, it can detect both WT and mutant p53. there is no problem with protein and transfer, i am doing the expt with L-132 cells (human lung epithelial cells)normal), i am getting other protein expression (bax, bcl2) with same sample. I am looking for apoptosis.
DO1 is a very very good antibody for p53 (but I prefer to use polyclonal p53 antibodies such as CM1 because it allows detection of all splice variants of p53.) Anyway you should see p53 protein with DO1 (at concentration1ug/ml) even at basal level in absecne of damage. How much protein do you load per well? below 10ug per well it would be very difficult to detect p53? and also how long do you expose your film? can you induce damage to your cells (doxorubicin (1uM ) , Actimonycin D (10ng/ml)) then extract protein 24h after treatment and see if you can detect p53 induction in your cells upon damage?
I am loading 40 microgram protein. I dont have positive control, but in my expt apoptosis in occurring, during apoptosis I should get p53 induction.
I would also expect p53 induction if you see apoptosis. do you see p21, mdm2 induction? how is bax ? if yes then your DO1 antibody is may be not working...
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