Problem with EGFP in translation study - (Oct/10/2006 )
Hi everyone!
I wonder if you could help me?
I perform translation study in mammalian cell culture. My construct include universal promoter of the vector, leader sequence of my gene of interest (which has a meaning in a translation efficiency), EGFP and FLAG tag. Using antibodies against FLAg I am able to obtain band of fusion protein of expected size in western blot - therefore I concluded that translation takes place in my culture. But EGFP signal is absent, when cells are observed under the fluorescent microscope.
Does anyone has an idea, what could be a reason of this discrepancy?
Thanks
L
I wonder if you could help me?
I perform translation study in mammalian cell culture. My construct include universal promoter of the vector, leader sequence of my gene of interest (which has a meaning in a translation efficiency), EGFP and FLAG tag. Using antibodies against FLAg I am able to obtain band of fusion protein of expected size in western blot - therefore I concluded that translation takes place in my culture. But EGFP signal is absent, when cells are observed under the fluorescent microscope.
Does anyone has an idea, what could be a reason of this discrepancy?
Thanks
L
Am I right? your fusion protein has two tags, an EGFP and AND a FLAG tag? or your constructs carry either an EGFP OR a FLAG? If they carry both you are able to predict molecular mass, and may estimate if both EGFP and FLAG are fused even with your anti-FLAG Ab;
I would first do a western blot against GFP. If the GFP is present in this blot, then it is inhibited somehow. Maybe the folding is incorrect and you need to make a spacer before the GFP. Or the medium inhibits the fluorescence somehow.
Thank you for reply.
Yes, my fusion protein includes BOTH tags. I do not have Ab against GFP, therefore I can't check it on western blot, but if it not include GFP, the band size would be much smaller. And fusion protein is recognized by FLAG Ab and Ab against my protein of interest (the whole fusion includes a part of my protein - GFP - FLAG, in this direction). There are also spacers of 7 and 4 aa between them.The only reason which I can imagine now is that folding of GFP is not proper in this fusion protein, or that it degradate very fast.
Any other idea?
L
I've noticed GFP fusion proteins seem to be less fluorescent that the GFP alone. Could you try flow cytometry it should be more sensitive that the microscope or turn up the gain on the microscope?
Ceri
HI
My fusion protein include: part of my protein (92 aa), spacer (9 aa), GFP (239 aa), spacer (4 aa) and FLAG (22 aa).
Could it be, that this protein folds much different than normal GFP, and therefore fluorescence can not be detected? And how to determine this?
L
My fusion protein include: part of my protein (92 aa), spacer (9 aa), GFP (239 aa), spacer (4 aa) and FLAG (22 aa).
Could it be, that this protein folds much different than normal GFP, and therefore fluorescence can not be detected? And how to determine this?
L
GFP fusion proteins do seem to have lower fluorescence or in many cases weak fluorescence. If u want to c the GFP fluorescence try using a stronger promoter. we have had similar problems till we switched promoters and compared them and started to use the stronger promoter.
even if u had a luciferase as a marker and comapred levels of luciferase alone and as fusion, u will find a difference. I am not sure abt the exact reason for this.
Even adding a tag to the GFP can in some cases decrease fluorescence. Reason for this could b interference with the folding of GFP.
Hi
I have one other idea and quoestion.
Maybe the reason, that I can not see the EGFP fluorescene is the low efficiency of transfection, and therefore transcription and translation (fusion protein can be detected by western blot, but it's amount in a single cell is too low to be detected by fluorescence).
How can I determine the efficiency of transfection, and which method (e.g. kit) could be the best solution (best efficiency)?
Thanks
L
do you have also a GFP expression vector (mock)? that and your GFP construct will do to test transfection efficiency independently; if you use a commercial transfection reagent follow the instructions for optimization, normally, testing different reagent: DNA ratios; if you have primary cells, transfection is always more difficult than cell line cells;
I have made a bunch of FLAG fusions and have found the FLAG antibody to be quite cross reactive, giving many bands on westerns of cell lysates. Thus, I'll ask this question first: Are you sure that the band that you got in your western of expected GFP-FLAG-fusion size wasn't present in an untransfected lane?
If you're sure of your western, then I second what Ceri has said about using flow cytometry. When I have done transients transfections, sometimes if the expression is low, I can't see it by microscopy but can using flow cytometry. You wouldn't need a GFP antibody for it either if you have a machine to use.
Just my insight, good luck,
Mountainman