For my experiment I will be treating HUVEC cell cultures with FGF-2 monoclonal nAb for 24 hrs. Since this cytokine works in an autocrine fashion, I was wondering if RT-PCR can be used to confirm that the FGF-2 protein has been completely neutralized? If RT-PCR is not a good choice, then please suggest alternatives?
IHi, Sandeep
No sufficient info about cells, time when you are collecting cells, and the specificity of the antibody you are using (specific only to FGF2, a growth factor). However, RNA and protein expression shoud be pursed together.
1) It would be necessary to evaluate the FGF2 and Ab specificity using other FGF family members. This is where you need to provide accurate info about what cell line, cancer cells? FGF2 shows autocrine signals in cancerous or pathological cells. However, it works as a paracrine signals in normal embryonic development and ES cell culture. There are many FGF family members, which often stimulate each other in a positive feedback loop.
2) The other point is that even if FGF secrete, bind to RTK and stimulate cells in a half way, it does not mean the gene expression (by RT-PCR) occurred. The genes of interest (GOIs) may not be expressed until some other genes are activated during cell differentiation in many tissues.
Therefore, try a small test run to see time-course effects of FGF2 for the analysis of the (GOIs) with negative control of other FGF member, and sham control. Then determine the duration of FGF treatment, and Ab addition.
3) If you are lucky, i guess, in most case, there won't be delayed expression of the GOIs because of other activation signals are yet to come in addition to FGF2.
Best wishes.
Dr.Lee, Thank you for your response. I have updated the question with specific details.
Hello
Kindly look at this links , hope help you
https://www.jstage.jst.go.jp/article/jrd/57/1/57_10-008K/_pdf
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5474279/
http://atvb.ahajournals.org/content/35/2/358
Good Luck
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