Can anyone suggest a protocol for measurement of p53 expression and ras after treating the cell lines with anti cancer synthetic compounds which induced apoptosis?
The simplest and informative approach would be to measure their activities (after transfection of p53 or Ras-responsive reporters into cells), levels of these proteins (by immunoblotting), and their activation status.
See the links for details:
1. http://www.ncbi.nlm.nih.gov/pubmed/11013253
2. http://www.ncbi.nlm.nih.gov/pubmed/12461788
The best way to measure p53 (both total levels and the activated forms via phosphorylation) and Ras would be via Western blot. Cell Signaling Technology has great antibodies that work well for p53 detection.
western blot is the best method to see the expression of p53, Phospho-p53 as well as ras protein. FACS analysis using fluorescence tagged antibodies is also done to see the changes of protein levels after drug treatment.
p53 reporter assay or western blot would be the best way to measure the activity or expression of the proteins
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Measurement of p53 levels (western-blot) and p53 expression (PCR analysis) is not sufficient for evaluating p53 activating status. The switch between the activated and the dormant status of p53 is mainly provided by its negative regulator
MDM2 (among other factors like MMX and p300). Additionally, p53 should be acetylated to be fully activated and protected from MDM2-degradating effects. Therefore, if your aim is to focus on p53 activation , you should consider a lot of integrated molecular parameters: p53, acetylated-p53, MDM2. For a reference, see : "Melatonin down-regulates MDM2 gene expression and enhances
p53 acetylation in MCF-7 cells" (J Pineal Res, 2014, 57:120).
bests,
Mariano
I used WB for both of these proteins, may I suggest you also try and find out if the p53 is mutated, if so, which mutation? Because you can find mutated p53 specific antibodies aswell. (not really sure how specific they are though...). Also, as said before, if you want to see if it's translation / post translation / transcription regulation - you should try PCR.
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