Technical Tips: Detection of Mutant and Wild-type p53
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1. Why might I not see wild type p53 without using radioactivity? Top
Endogenous levels of wild-type p53 are low and may be difficult to detect.
2. What ELISAs are available? Top
Mutant p53 can be detected using our mutant p53 ELISA kit, Cat# QIA03. Both mutant and wild-type p53 can be detected with our rapid format pantropic p53 ELISA kit, Cat# QIA26. Complete instructions are provided with the kits. (Note that endogenous levels of wild-type p53 may be below the level of detection by ELISA.)
3. How do I detect immmoprecipitated p53 without using radioactivity? Top
When doing an IP/Western do not use an unconjugated mouse primary antibody for detection in the western blot. The anti-mouse second step will detect the heavy chain from the mouse precipitating antibody, and this 56 kDa heavy chain migrates close enough to p53 to mask its presence. Use instead our western blotting kit, Cat# WBB08. This kit uses a biotinylated anti- p53 antibody which is detected with a streptavidin conjugate and thus avoids detection of the precipitating antibody. Cat# PC35 can also be used in the western blot step to detect immunoprecipitated p53. Use the sheep p53 antisera as the primary antibody as described in our western blotting protocol, Fatty acid-free BSA should be subtituted for milk, which contains biotin and can increase the background. Detect with an enzyme-linked anti-sheep antibody or use the biotinylated secondary antibody included in the PC35 kit along with streptavidin-horseradish peroxidase (Cat# OR03L) or streptavidin-alkaline phosphatase (Cat# OR04L). When using OR03L or OR04L, incubate with 0.2 m g/ml enzyme conjugate for 1 hr at room temperature in TSBT, 0.5% BSA. Wash 3 times and add substrate as detailed in the detection system protocol. Expected results:
• Only mutant p53 should be present in the lanes immunoprecipitated by OP29.
• Only wild-type p53 should be present in the lanes precipitated by OP32/OP33.
• All p53 in the sample should be present in lanes immunoprecipitated by OP03/OP43.
4. What antibodies are avaiable? Top
Immunoprecipitate sample(s) with one or more of the following antibodies using our standard protocol using separate tubes for each antibody:
Mutant specific, Cat# OP29 (human, mouse, rat, other)
Wild-type specific, Cat# OP32 (mouse) or OP33 (human)
Pantropic (detects both mutant and wild-type), OP43 (human) or OP03 (mouse, rat)
When radiolabeling wild-type p53 with [35S]methionine, incubate for 1 hour. Boil the protein A and/or Protein G beads in sample buffer for 3-5 min, centrifuge briefly to pellet the resin then separate the eluted proteins by gel electrophoresis. Detect using autoradiography (for radiolabeled samples) or western blotting (use Cat# WBB08) and chemiluminescence.
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