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Difficulty in expressing fusion protein - (Jan/27/2012 )

I am trying to make a fusion protein. My model organism is zebrafish. Initially I tried to express my protein with Mcherry at its N terminal. I could see the expression of Mcherry in my wild type fusion protein. However, when I made a mutant fusion protein, I couldnot see any expression of Mcherry. Then I repeated the same procedure with GFP as a fusion and got the same result. I tried to check if the protein is expressing by western, but the antibody doesn't work. I tried using a GFP antibody, and got a thin band with my Wild type fusion protein but the size of the band was same as that of GFP alone.

Can anybody help out?



verify that both the proteins are aligned and not out of frame. try the other way round, with gfp in C terminus. or simply use a small tag to your protein of interest instead of gfp and use ab against tag to detect the protein.