Hello all, Instead of looking for immunofluorescent of EYFP (as we have some problem), we are planning to do western blot, for EYFP that is attached with human channel rhodopsin 2 and AAV5 (plasmid). After the transduction, we would like to do western for EYFP using anti-GFP antibody, do you think, this will work? As I am running 10 - 20% gradient gel (commercial), do you think, EYFP will denature? or alternatively, if it works, will EYFP migrate to its own size (25-35 kDa) or will that bound with channel rhodopsin 2, therefore, I have to look at 170 kDa instead of 25-35 kDa? All your answers are highly appreciated and would help me moving forward. Thanks.
If you fuse the gene for EYFP with your rhodopsin gene on the plasmid then it should make a single protein that is a fusion between the two. Thus it will migrate at the kDa of rhodopsin+EYFP. EYFP is similar to GFP except for a few point mutations so if your antibody is a polyclonal it may be detected with the anti-GFP antibody. Most GFP antibodies will work for GFP variants. If you are running SDS page before Western, the antibodies should still detect your protein fusion.
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