Currently, I want to check the function of protein A in fibroblast cell line and pancreatic cancer cell lines. Is it possible in theory?
For example, I want to check the expression of protein A in fibroplast cell line and different pancreatic cancer cell lines with different gene mutations. In my hypothesis, I guess that protein A is over expressed in pancreatic cell lines, and promote carcinogenesis. I plan to check it by WB and IF. Can someone tell me whether is it possible? Because they are different cell lines, so I confused whether I can do it?
As the previous comment tells, you have to check how amount of protein A is expressed in the cancer cells and normal fibroblasts in the steady state. Moreover, you have to keep in mind that some kinds of mutations cause conformational change, which makes antibodies unable to recognize anymore.
To investigate into the functional assay, you have to perform both the overexpression and knockdown of the protein A in several cell lines. The function of the protein is often different depending on the cell context (in this case "PDAC vs fibroblasts"). That is why you have to check in terms of cell migration, proliferation assay as well as tumor formation in immunocompromised mice etc.
I agree with both comments. Moreover, pay attention to controls, especially when trying to compare different cell lines (for example, is the normalization house keeping protein expressed to the same extend in both cell lines, otherwise the interpretation will be fallacious); try to use different antibodies to limit effect of epitope changes; pay attention from results generated in IF and WB with the same antibody (you may reproduce artifacts), and so on.
For functional assays, K.O. or siRNA silencing is clearly a way and corresponding overexpression is a must (may help to investigate compensation mechanisms) and screen several assays first and if possible, use different versions of the one that works.
I hope it well help.
Best.
seb.
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