FLAG-GFP-tagged protein problems - (Dec/02/2008 )
Hello all,
I have cloned my cDNA of interest in a expression vector to express it in mammalian cells as a 3xFLAg-GFP-S protein fusion. The protein is quite big, is 200 kD. Everything was OK, the construct, no mutations, is localizing properly when I do IF, I can detect it by WB anti-flag and anti-protein...I have stablish stable cell lines and I already have monoclonal populations properly expressing it by IF, my problem is that a part from my band at the correct size for the expressed protein I also see a band at the size of GFP qhen I do anti-flag WB.
I don't know why, could it be some degradation product? is it normal? any help is more than wellcome, thank you
I have cloned my cDNA of interest in a expression vector to express it in mammalian cells as a 3xFLAg-GFP-S protein fusion. The protein is quite big, is 200 kD. Everything was OK, the construct, no mutations, is localizing properly when I do IF, I can detect it by WB anti-flag and anti-protein...I have stablish stable cell lines and I already have monoclonal populations properly expressing it by IF, my problem is that a part from my band at the correct size for the expressed protein I also see a band at the size of GFP qhen I do anti-flag WB.
I don't know why, could it be some degradation product? is it normal? any help is more than wellcome, thank you
possibilities:
1. degradation, not necessarily from preparation but ubiquitinylation or SUMOylation, is possible;
2. splice variant
3. co-transfection with an incomplete version of your gene of interest
do you detect the phenomenon in others of your clonal cell lines?
Hi,
Thank you for suggestions, I do observe this band in other clons, I also have lines stably expressing only FLAG-GFP tag and when I run a gel comparing samples the band I obtain is the same size as FLAg GFP. I was wondering if this could be a cleavege of the tag that is N terminal, while I lysate cells and run gels. This band is not as strong as the FLAG-GFP alone but it is always there.
When I do IF anti-GFP I never see background suggeting that some cells are not expressing properly my protein of interest, the IF looks like the normal endogenous protein.
Thank you again for your help
I've observed with a couple different GFP tagged constructs that I obtained a similar cleavage product about the size of GFP. I think it's a pretty common occurrance and I wouldn't worry too much about it.