GFP Fusion protein - (Dec/18/2006 )
Hi every body
My friend and I need some help.
We use GFP fusion topo TA expression vector to express a protein coupled to GFP in C ter.
We have a KOZAK sequence in our forward primer. PCR product is in frame with GFP (check by sequencing).
We do not detect GFP fluorescent by microscopy . Neither GFP nor interest protein by WB .
We also use YFP fusion topo TA expression vector to express the same protein coupled to YFP in C ter. The forward primer is the same. Our protein is detectable by microscopy because of a strong YFP fluorescence .
Is there so who can help us understanding this problem?
Thanks in advance
Are u using the same promoter for both constructs, GFP and YFP?
Yes, both contain CMV promoter
Stupid question maybe, but are you sure you're using the right filter for excitation/emission?
Have you tried to express just GFP in your cells?
I would try vairus's suggestion. Transfect only GFP and check if u get fluorescence.
transfect both YFP and GFP side by side. YFP should serve as a positive control for ur transfection.
This could shed some light on the problem.
Thank you for your help.
We used the right filter for GFP, and when we transfected GFP or YFP alone, we had fluorescence... but we found the problem: to obtain a strong fluorescence, we must transfect our cells with 2.5x more vector than the manufacturer's protocol.
so did you figure out why your protein is not expressed?