What isoforms will my p53 antibody detect?

Numerous p53 splice variants have been documented in the literature. They arise from alternative splicing of p53 transcripts and result in multiple isoforms. UniProt for the human sequence (UniProt P04637) lists 9 isoforms produced by alternative promoter usage and alternative splicing and there may be more, depending on the model. Some of these splice variants are associated with polymorphisms or mutations of the p53 genes. Isoform 1 (43.6 kDa) is predominant. However isoform 2 (37.8 kDa) and isoform 3 (38.5 kDa) are also well documented. The combination of p53 cleavage and alternative splicing allows for a diverse size-range of p53 proteins, however, the study of p53 degradation has focused largely on the canonical full-length isoform 1. Also note that the proline-rich region of p53 slows the migration of p53 in SDS-PAGE gels, increasing the apparent molecular weight of the p53 isoforms that express it. [see M.P. Khoury and J.C. Bourdon. (2010) Cold Spring Harb Perspect Biol. 2(3):a000927, Review (PMID: 20300206 https://www.ncbi.nlm.nih.gov/pubmed/20300206)]. Thus the predicted molecular weight of isoforms listed in the various databases are about 10kDa smaller than the observed molecular weight.

Generally speaking, most commercially available antibodies react with amino terminal regions of human p53 protein. Several isoforms are missing this region, such as isoforms 4, 5, 6, 7, 8, and 9. We do not have empirically derived data documenting isoform-reactivity.

Both the p53 (DO-7) Mouse mAb #48818 and the p53 (DO-1) Mouse mAb #18032 are produced by immunizing animals with recombinant human wild type p53 protein expressed in E. coli. These antibodies are made from commonly used clones that are validated at CST. A web search for these DO clones will show other sources reporting the epitope residing within the first 50 amino acids. Because we have not mapped the binding epitope for these antibodies at CST, we are unable to guarantee they will recognize all p53 isoforms.

The epitopes of the polyclonal p53 Antibody #9282 have been mapped. The antibody binds to amino acids within the first 80 amino acids of human p53. This antibody will recognize the canonical full-length isoform 1 and is predicted to bind isoforms 2 and 3, which retain the entire amino terminus region bound by #9282. Based on sequence, it is possible that #9282 may also bind isoforms 4, 5, and 6. These isoforms retain amino acids 40-80, which contain some of mapped epitopes for #9282. Based on sequence, #9282 should not bind isoforms, 7, 8, and 9. These isoforms do not contain amino acids 1-132, therefore they lack the entire #9282 binding region. 

The binding region of the p53 (7F5) Rabbit mAb #2527 has been mapped to the amino terminus region of human p53 protein. It will recognize the canonical full-length isoform 1 and is predicted to detect isoforms 2 and 3.

The p53 (1C12) Mouse mAb #2524 is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser20 of human p53 (also the amino terminus region of human p53 protein). It will recognize the canonical full-length isoform 1 and should detect isoforms 2 and 3.

The p53 (E9B5W) Rabbit mAb #30313 was produced using a synthetic peptide antigen corresponding to residues surrounding Ala347 in the tetramerization domain of human p53 protein. This is currently our only total p53 antibody that has an epitope that does not reside in the N-terminus. Since the epitope resides in the tetramerization domain, the p53 (E9B5W) Rabbit mAb #30313 will only recognize monomeric p53 under denaturing conditions. #30313 binds isoform 1 and is also predicted to bind isoforms 4 and 7.

 

Last updated: September 12, 2024