NIH3T3 protein expression - it isn't working (Jan/20/2006 )

Hi All-

I am trying to create a NIH-3T3 stable cell line, overexpressing my protein of interest.

I just finished cloning the cDNA into my expression vector and have been testing transiently transfected 3T3's for expression of my protein with western blot, before I begin the long process of creating a stable line.

When I transfect a pEGFP vector alone I see expression of EGFP by microscopy and western, but cannot detect my expression of my Protein-IRES-EGFP vector in transiently transfected cells.

I checked the sequences and they are all perfect, so that can't be the problem. I have also tried optimizing the transfection under various conditions and in all of them I see EGFP expression but not my protein of interest. (well except for one mutant construct once, but I wasn't able to reproduce it this week)

Does anybody have any ideas as to what I should try next to troubleshoot? I have always used just a 24 hr post transfection timepoint, so I was thinking that maybe this protein may have a slow expression curve or something? any other ideas?

Thanks for your help,

Mountainman

might be.. if i'm correct 48h is usually considered as a peak of protein expression after transfection.. maybe you could try to wait a bit longer?
when i transfect GFP (EF1alpha promoter) into 3T3, after 24h i can see only w very few green cells, much more after 2 days though.