EGFP detection - (Aug/11/2005 )
Hi
I was trying to produce some translational fusions of a particular gene with EGFP. Before I did a flow experiment, I wanted to check (by western) whether my transfected cells express EGFP. I purchased a GFP antibody (mouse mAB). But in westerns, I see a positive GFP signal (~25KDa) only in cells transfected with vector alone and not in cells which have gene of interest translationally fused to EGFP.
Has someone encountered such a problem? If so would you be willing to suggest a remedy to my problem?
Thanks
AZCBIO
I was trying to produce some translational fusions of a particular gene with EGFP. Before I did a flow experiment, I wanted to check (by western) whether my transfected cells express EGFP. I purchased a GFP antibody (mouse mAB). But in westerns, I see a positive GFP signal (~25KDa) only in cells transfected with vector alone and not in cells which have gene of interest translationally fused to EGFP.
Has someone encountered such a problem? If so would you be willing to suggest a remedy to my problem?
Thanks
AZCBIO
I am also working with EGFP plasmid. In my case, the inserted coding sequence connects to the EGFP sequence and they together produce a fusion protein, which is larger than either one of them. If your construct works the same way, the GFP band would apprear at a different size (=protein of interest + 25kDa) for cells transfected with your gene of interest. And, if the fusion protien is a large one, say > 100 kDa, you may need to increase the transfer efficiency of WB in order to detect it.
Hi
Thank you for your response. I had almost given up hope!
I did consider the possibility of a different sized product, however, the problem with my WB is that there is simply no band in my transfected samples. Although, the vector control has a band detected by EGFP antibody, the rest of the WB is simply blank. I will try increasing the transfer efficiency anyway, to check whether that does the trick!!
Thanks Again
AZCBIO
Hi
I was trying to produce some translational fusions of a particular gene with EGFP. Before I did a flow experiment, I wanted to check (by western) whether my transfected cells express EGFP. I purchased a GFP antibody (mouse mAB). But in westerns, I see a positive GFP signal (~25KDa) only in cells transfected with vector alone and not in cells which have gene of interest translationally fused to EGFP.
Has someone encountered such a problem? If so would you be willing to suggest a remedy to my problem?
Thanks
AZCBIO
I am also working with EGFP plasmid. In my case, the inserted coding sequence connects to the EGFP sequence and they together produce a fusion protein, which is larger than either one of them. If your construct works the same way, the GFP band would apprear at a different size (=protein of interest + 25kDa) for cells transfected with your gene of interest. And, if the fusion protien is a large one, say > 100 kDa, you may need to increase the transfer efficiency of WB in order to detect it.
Is GFP on the N- or C-terminus of your protein of interest? If it's on the C-terminus are you sure it's in frame with the rest of the protein. I would have thought the easiest way to check for expression would be to look under a fluoresence microscope or by flow cytometry if you have access to the equipment.
All the best,
Ceri
Hi
My EGFP is on the C terminal and is in frame. I have looked under the fluorescence scope and I do see good fluorescence. However my westerns have been the problem. And I have no idea why. I have tried using antibodies against my protein of interest as well. However as they are deletions, I still can't detect two of my fusion constructs.
Thanks you for the advice though!
AZCBIO