Hi all,
I have made an NLS-superfolding GFP (sfGFP) driven in pcdna3.0 and am trying to express it in L929 cells - mouse fibroblast line. Unfortunately, I am not having any luck. The construct expresses great in HEK293 cells and shows nuclear colocalization by immunofluorescence. I am using the GenJet transfection reagent to transiently express my sfGFP plasmid (24 hrs post transfection check expression). I have a control mCherry plasmid also on a pcdna3.0 backbone that expresses fine in my L929 cells so they are "transfectable."
I should clarify that I have just tried to check expression by IF and ICC and am in the process of performing westerns to check expression.
Any thoughts on how to troubleshoot this problem?
Best,
You have all proper controls. So it is very interesting to know where the problem is. Do you have access to flow cytometer? If so, I would suggest to check with that. You may find at least a shift of histogram for gfp though the expression is too small to detect by microscopy. Use the Hek cells as a positive control. Good luck
It seems you don't have an issue with the construction of the plasmid , however, you may have a problem in sfGFP expression and/or stability in the L929. Check if your protein was degraded.
Try the western blot experiment to check the expression of sfGFP.
Maybe, GFP was quenched in the L929 cells.
Tried the western blot experiment but could not detect the protein. The transfection efficiency of this cell line is ~60 % so it is possible that I just did not pool enough samples together to get a signal.
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