Hi,
I am working with A549 cells. I want to increase the expression of p53 after being treated with several inhibitors, but:
1. I got double bands.
2. I got a band in the control (untreated) group.
In several papers reference that A549 cells have p53 wild type. Also, the result of their western blot data did not show bands in the untreated group.
What can be the problem? Or there is any explanation to this behavior?
The simplest explanation is that you have cells that have undergone a mutation in one of the allele in p53 and you detect a protein band in control extracts because the mutant p53 is stable and detectable. I would be more interested in learning whether the p53 can be activated in your cells after treating with a DNA-damaging agent (as determined by transcriptional activation of certain p53 target genes).
Hi Veronica,
A549 cells have a deletion on the CDKN2A locus that harbors p16 and p14ARF genes. Without p14ARF, MDM2 levels are high, which keep p53 low. Even though p53 levels are low, there is still a basal level of the protein. A band signal will depend on how long your blot was exposed.
Also, some antibodies for p53 can be very "dirty" (low specificity, high cross reactivity). So a good idea is to knockdown p53 to use as a negative control. This way you make sure you are looking at the right band.
Recently I used an antibody from Cell Signaling that was pretty clean (#2524S) (although a bit expensive).
Best of luck!
I think that both answers, (the one of Divaker and the one of Jacqueline) are possible answers to the mistery. A549 cells may show a low basal presence of p53 if the method to detect it is very sensitive. The second band may be due to mutated p53.
Excellent information. So far, I was also expecting some changes in the expression of p53 after enhance the expression of EGFR but I found some literature that suggest that EGFR phosphorylation will not modified the expression of p53. Also I found that my cell line had mutation on p53 also.
Thank you so much to three of you for your comment, with them, I found more information that help me to understand my result.
Best of luck in your currently studies.
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