HOW TO detect target gene - after the transfection (Apr/24/2004 )

hi,
I am in a difficult situation now,that is to say,I have transfected my target gene into the cells using EGFP as a reporter gene. My question is how to detect the expression of my target gene addition to the observation of EGFP.By the way,I have no antibody to target protein.
Thank you very much!

Would a protein gel help? Maybe not if amounts are too low. System perturbation analysis might be a better approach.

How about using Flow, RT-PCR or qRT-PCR? But I think the better method is to do a WB using antibody against Tag or directly against protein of your interest.

Good luck,


Alex

Thanks a lot! I will have a try according to your suggestions.

Another question is : Can I do W-B using antibody against the reporter gene EGFP though it can be detected by the eye?

I think so if you have the anti-EGFP. What system are you working on? What kinds of equipments are available in your lab? If possible you can staining you cells with anti-GFP and do immunohistory staining. or More straight-forward see them under immunofluence microscope by you naked eye and take a picture. Or use EM so that you also see the cellular localization of you protein.

Anyway it is up to you: what equipments are available and which technique you are more cofindent with?


Hope this help,


Alex

hi, alex_osu3

Thank you for your advice. And the equipments are no problem!