way to confirm that my protein is active - (Dec/14/2005 )

Hi everyone,

I have put a cDNA of my protein of interest just beside a good promotor and I have put this into human myoblasts cells. I saw, on western blot, that I have an overexpression of my protein, and that is good.

Now, I want to know if the protein that I have overexpressed is fully actived, i.e. if it has the same function as the protein that is normally expressed by my cells. What sould I do?

Thanks,

what is th function of your natural protein? exp should be function related...

hi,

My protein of interest is myogenin. This protein is a member of a myogenic regulatory factor. Myogenin binds to some promotor sequence and allow the transcription of specific gene.

Hi!

You could try to do reporter gene assays, or just examine if target genes are transcribed after transfection. Should give you an indication about the function.
Cheers

hi i agree woth bomber.
if you have a tag at n or c ter of your protein overexpressed you may purify it and see in transcription assays if you have same levels as well using flagged as the wt one.