CM1 and Sapu polyclonal antibodies are highly specific and with high affinity for p53. DO1 is a great antibody but it recognises an epitope localised between amino-acid 20-25 which is phosphorylated and it is published that phosphoryaltion of ser20 inhibits binding of DO1. Moreover, the epitope 20-25 is lost in the other p53 isoforms due to alternative splicing. If you have to use a mouse monoclonal antibody, use DO7 which has the same epitope as DO1 but its binding is not inhibited by phosphorylation of ser20.

cheers

An important means to confirm the results

Elisa are in my view the best way to quantify p53 if you know the epitope of the antibodies that you are going to use. I use CM1 (Rabbit polyclonal antip53 that recognise several epitopes in the N-terminus and C-terminus of human or mouse p53) and then I use sheep polyclonal anti-p53 antibodies raised against recombinant p53 followed by a secondary anti-sheep HRP conjugated antibody from jackson immunological with low cross reaction with rabbit or mouse IgG. As a positive control and to have absolute quantification, I would suggest to use purified recombinant human p53.

Many epitope of mouse monoclonal p53 are masked by phosphorylation, ubiquitinylation, sumoylation, neddylation, acetylation, methylation etc. Morevoer human p53 gene express at least 12 different p53 proteins (p53 isoform) due to alternative splicing, alternative initiation of translation and alternative promoter usage. WE and others have demosntrated that p53 isoforms play key roles in the cell cycle progression, cell death, sensecence, ageing, differentiation, etc. The modualte gene expression in a p53 dependent and independent manner. some of the isoforms are localisaed int he cytoplasm other shuttle between the nucleus and the cytoplasms. it is possible to identify p53 isoform proteins by using a panel of monoclonal antibodies and siRNAs. please have a look at my publications on p53 isoforms and do not hesitate to contact me if you want more informations.

Yes, this is the best way to assure the level of p53, however, p53 phosphorylation status would be more important and if it is phospho ELISA, there is no question about it. Thanks.

CM1 and Sapu polyclonal antibodies are highly specific and with high affinity for p53. DO1 is a great antibody but it recognises an epitope localised between amino-acid 20-25 which is phosphorylated and it is published that phosphoryaltion of ser20 inhibits binding of DO1. Moreover, the epitope 20-25 is lost in the other p53 isoforms due to alternative splicing. If you have to use a mouse monoclonal antibody, use DO7 which has the same epitope as DO1 but its binding is not inhibited by phosphorylation of ser20.

cheers

Thanks everyone - and for measuring mouse p53 does anyone have a 'best' antibody ?

the best antibody for mouse p53 is CM1 (rabbit polyclonal p53 antibody), although it was raised against human recombinant p53, it recognizes several epitopes within p53 which are also present in mouse p53 protein. the other good antibody is mouse monoclonal 1C12 whose epitope exists in human and mouse p53 (around ser20 in human p53).

Thanks you Jean-Christophe, just two hours ago I used 1C12 in a western blot with great results. Thanks, will also look into CM1.

1c12 will not see all isoforms of mouse p53 but it is a good antibody to see p53 protein containing the N-terminus of p53

if you need cm1, I can send you an aliquot

That would be really great Jean-christophe ! If this is still OK, I will email you. Thanks.

Hi Jean-Cristophe (et al). Please see below a comparison of CM1 and IC12 via western blot in a trial of p53 siRNA. Thanks so much for the antibody! Do you have a suggestion as to the identity of the additional band at 110kDa - just a dimer ??

Although specific, WB has some limitations as they can't recognize conformational or native epitopes, ELISA can detect both linear and conformational epitopes. WB is more qualitative than ELISA whereas ELISA is more quantitative than WB. If possible, I would recommend both of them.

HI Bobbi,

Nice western! if I understood well, you transfected increasing amount of siRNA specific of p53 mRNA in your BV2 cells. ctrl is loaded on right hand side of both blots ( ctrl "No": is it mock trasnfected or is it trasnfected with a non relevant siRNA?) Several bands are visible with CM1, one band at 110kD that does not disappear byincreasing siRNA p53, a doublet at 53kD (well visible at 1nM and 0.3nM, and a 45kD band (which is visible with1nM, 0.3nM and 0.1nM, less visible with 3nM but not visible after 0.01 or No). only one band is detected with 1C12 (green). CM1 is rabbit polyclonal which recognises several epitopes into p53 (N-term and C-term domain of p53). so that CM1 detects all p53 protein isoforms. The mouse monoclonal1C12 recognises only one epitope around amino acid 20 in mouse p53 which is not common to all p53 protein isoforms.

Have you done the blot with only the red secondary antibody that you use for CM1? (are the bands at 110 and 45kD specific of CM1 or due to secondary antibody)

Have you repeated the blot with different extracts since that one?

do you have a very long exposure of this blot?

Since p53 encode several p53 protein isoforms due to splicing and internal promoter usage, it would be very interesting to confirm that the 110 kD bands, the doublet at 55kD and 45kD band are p53 protein isoforms detected by CM1. (1C12 does not detect p53 protein isoforms because they do not contain 1C12's epitope). the doublet at 55kD and 45kD band are consistent with what we have for p53 in other mouse cell lines, We see additional bands after longer exposure , doublet just above 45kDband and we detect also a band at 28kD. However, we do not detect bands at 110kD.