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[Pranav2408]

Hi,
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?

Thanks

[scolix]

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.