Differences in epitope accessibility of p53 monoclonal antibodies suggest at least three conformations or states of protein binding of p53 protein in human tumor cell lines.
The p53 tumor suppressor gene is deleted or mutated in over 50% of human tumors. Mutations frequently extend the half-life of the p53 protein; and a high level of nuclear p53 expression, detected by immunohistochemistry, has been used to predict the p53 status of tumors. We compared the sensitivity and reactivity of five frequently used, commercially available monoclonal antibodies (1801, DO1, DO7, BP53.12 and 421) in immunoblot and immunofluorescence assays, and found that results differed among the antibodies. Comparison of immunoblot analysis of denatured nuclear and cytoplasmic p53 protein were consistent with antibodies DO1, DO7 and BP53.12, each of which generated a strong specific signal in both cell fractions. However, in situ analysis demonstrated that although all antibodies recognized nuclear p53, only BP53.12 and 421 recognized p53 protein in the cytoplasm. In addition, 1801 produced a signal in p53-negative tumor cell lines. Differences in situ among the antibodies were probably due to the accessibility of their respective epitopes and suggested that nuclear and cytoplasmic p53 either have different three-dimensional conformations or are bound to different proteins. A third p53 protein conformation was also suggested by the observation that only two of the five antibodies (BP53.12 and DO7) detected induced levels of p53 in situ following exposure to ionizing radiation. In summary, except for the fact that DO7 does not recognize cytoplasmic p53 in situ, we found it to be the most specific, versatile, and reliable antibody. We conclude that the p53 antibody of choice depends upon the specific goal of a study and the method used to detect this protein.
Danks
M K
MK
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101, USA.
Whipple
D O
DO
McPake
C R
CR
Lu
D
D
Harris
L C
LC
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D018985:Epitopes, B-Lymphocyte / Q000276:immunology*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015151:Immunoblotting; D011487:Protein Conformation; D011839:Radiation, Ionizing; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D018985:Epitopes, B-Lymphocyte; D016159:Tumor Suppressor Protein p53