Antibodies to murine p53 protein work really well in immunoblotting (see our papers in links below).

http://www.jbc.org/content/276/1/298.long

Article Increased Expression of Ifi202, an IFN-Activatable Gene, in ...

Hi Georgia,

There are many anti-p53 Abs that are able to recognise mouse p53 (see also Article SMIFH2 has effects on Formins and p53 that perturb the cell ...

Article Proteomic Analyses Uncover a New Function and Mode of Action...

I wonder whether there was no (specific) signal also in the positive control as the levels of p53 could be below the detection limit of your Abs in normal unchallenged (mouse) tissue.

In other words, it might well be that your Abs are ok.

Good luck!

Metello

If the tissue contains low endogenous levels of p53 protein, try IP-western using agarose-conjugated anti-mouse p53 for IP and detect the IPed p53 by immunoblotting.

Great, thank you. I'll give it a try

In our hands, p53 (DO-1, sc-126) works very well with a low cost. Thanks.

I agree with Jian, DO-1 antibody works very well with human and mouse p53 protein (though the quality might be supplier-dependent).

For more p53 antibodies you can also check here.

http://p53.free.fr/p53_info/p53_monoclonal_antibodies/p53_Mab.html

Thanks all for your help. I have tried the DO-1, both the older version sc-6243, and the replacement antibody that SC are making, sc-126. Neither of these antibodies gave me a band close to 53kDa. The mice tissues are from mice that have undergone interventions in which we expect to see a stabilisation of p53 (so not basal conditions). We also see induction of p53 target genes on the mRNA level...very annoying as I have tried around 6 different antibodies at this stage and none seem to be specific :(

Hi Georgia, do you have positive or negative controls in your detection experiment of p53. Your sample may have the isoforms of p53 or the modifications on p53, which caused you got the bands not close to 53KDa. Thanks.

Hi Jian Lui, I used a positive control in my most recent blots (cell line over expressing p53) with the DO-1 ab, and received no band at all present in the + control, but I did receive bands at around 65 and 25 kDa on my other samples.

I guess isoforms is a possible explanation but I would still expect to see some of the full length protein on the membrane right?

Thanks anyway for your help

Hi Georgia, you may want to have one confirmed negative control, such as p53 knockout cells or tissues. p53 can be detected usually. Good luck.

Dear Georgia,

While the DO-1 Abs does not react against p53 of mouse origin, it is unlikely that all the other five Abs that you have tested are human specific.

Given that both the tissue samples and the positive control, the latter being a cell line, show no specific signal (the main p53 band is 53 kDa as the name indicates), I would then guess that there is a general problem with sample preparation and/or the SDS-PAGE.

Thus, it could be useful to probe the same membrane with unrelated Abs. For example, if lysates are degraded or SDS is missing, most proteins would show an aberrant migration pattern and size.

Hope this helps.

Best,

Metello