Hello everyone,
I have one research problem. I have to separate p53 (MW 53 Kda ) from total protein extracted from cancer cells and I have to study it by LC-MS. I have separated the 53 Kda protein from total protein using gel electrophoresis. But the LC-MS studies showing the results for alpha tubulin( MW 50 Kda).
I want to know how can I separate only p53 from total protein and use it for LC-MS.
Thanks
Sachin
Many p53 questions on this topic have been asked on RG already. If you go to the search box and use the drop down menu to get to "question" you can then type in the key words for your question. Here's an example:
Jillian Cathcart 12.41
Stony Brook University
Why when I overexpress p53 do I observe a decrease in the amount of chromatin precipitated with an anti-p53 antibody?
I am performing chromatin immpunoprecipitation assays (ChIP) in which I transfect HT-1080 cells with vector control or p53. When I use an anti-p53, I consistently pull down less DNA in the p53 overexpressing cells when compared to the vector control cells. This is even observed at the p53 binding site in the p21 promoter, which should function as the positive control for this experiment. We know the transfection efficiency is high (at least 75%) and the cells are not dying. Are there any other reasons this could occur?
Why when I overexpress p53 do I observe a decrease in the amount of chromatin precipitated with an anti-p53 antibody? - ResearchGate. Available from: https://www.researchgate.net/post/Why_when_I_overexpress_p53_do_I_observe_a_decrease_in_the_amount_of_chromatin_precipitated_with_an_anti-p53_antibody [accessed May 10, 2016].
Hello Adam,
I did not do any IP yet. Can you please provide protocol for it.
Thanks
Here is a kit for p53 immunoprecipitation, with protocol.
http://www.chromotek.com/fileadmin/user_upload/pdfs/Manuals/p53-Trap_A_kit__manual.pdf
I think you need a Two-dimensional Gel Electrophoresis, have a look at these two papers and good luck
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