We have tried to localize the p53 and p21 in MCF-7 cells after treatment of drug. We found that p53 located around the nucleus (hoechst stained) but very rare inside signals. Again in negative control we got cytoplasmic p53 and p21.is there any problem in antibodies? (FYI: both the antibodies are monoclonal). Also we need suggestion regarding good antibody which locate the active p53 and p21.
I agree with Oscar. Since you don't know exactly how your drug is working in the cells, it would be in your best interest to use a positive control for DNA damage that will strongly elicit a response. Then compare this to your drug treated cells using the same times point for comparison.
Are you trying to detect total p53 or a specific phosphorylated form? I would recommend detecting phospho p53 S15. This site is phosphorylated by p38 following cellular stress and this phosphorylation event helps stabilize p53.
You want to detect this by immunofluorescence. Have you considered to perform a cytoplasmic versus nuclear Western blot? I have antibodies that detect phospho and total p53 as well as p21 very well by immunoblot. I'm not certain how they perform in ICC.
As a first step I would suggest exposing your cell line to ultraviolet light (UVL) in the mutagenic range (230-300 nm) so to elicit DNA damage, DNA repair and nuclear localization of p53. It's better to do it side by side by covering your control cells with something opaque. Does your drug elicit DNA damage or repair? If not, try 5-Aza-D-2-cytidine, it should cause DNA repair.
I agree with Oscar. Since you don't know exactly how your drug is working in the cells, it would be in your best interest to use a positive control for DNA damage that will strongly elicit a response. Then compare this to your drug treated cells using the same times point for comparison.
Are you trying to detect total p53 or a specific phosphorylated form? I would recommend detecting phospho p53 S15. This site is phosphorylated by p38 following cellular stress and this phosphorylation event helps stabilize p53.
You want to detect this by immunofluorescence. Have you considered to perform a cytoplasmic versus nuclear Western blot? I have antibodies that detect phospho and total p53 as well as p21 very well by immunoblot. I'm not certain how they perform in ICC.
Thanks both of u. Yes Koschembahr I will definitely try with anti phospho p53 S15 and total p53 and p21 by ICC and immunoblot.
Can you suggest any method to analyse whether p53 (as a transcription factor) is transcriptionaly active or not ?
MCF7 should be wild type p53 and have virtually no p53 staining at all. Perhaps you should use several types of controls: a breast cancer cell line with mutant p53 that constitutively over expresses p53 in the nucleus AND as suggested by the others a treatment that can induce wild type p53.
Since both antigens appear cytoplasmic where they should not be, the straight forward answer is that you see an artifact. Again, using a cell line with nuclear p53 may help you to get your staining technique in order. Before you have not settled this, there seems to be no need to go for the next step and look for fancy antibodies detecting activated p53.
Controls are 50% of your experiment, 40% is labeling, the rest is your actual experiment...
p53 is retained in the cytoplasm in MCF-7, as it is in many neuroblastomas, maybe as a way to allow these cells to cope with wild-type p53. (Moll et al, Mol Cell Biol. 1996 Mar;16(3):1126-37.
U2OS treated with a DNA damaging agent would be a nice control to see nuclear staining of p53. DO-1 is a great antibody for that.
In my experience, anti-p21 (SMX30 from BD) is also very good for immunostaining.
A reporter assay using p21 promoter-luciferase (or RGC-luciferase) would be a very simple and standard way to determine if p53 is transcriptionally active.
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