I am trying to identify a protein with very low expression which is labelled with H2B-GFP. I tried to identify GFP expression using anti GFP antibodies. however, no bands observed in western blot and also no expression observed after immunofluorescence staining (using secondary antibody conjugated with FITC) of mouse tissue sections. GFP insert is on correct reading frame to that targeted gene. can anyone provide some suggestions for me
Hello
Are you sure of the protocol you used ?
Article Promiscuous Expression of H2B-GFP Transgene in Hematopoietic...
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If the protein is expressing GFP, no matter how low, you should be able to verify GFP expression in the FiTC channel just by flowing the cells without any antibody stain. Using a non-GFP control would help to visualize even a slight shift in the peak (if any).
Which terminus of your protein the GFP is present ie N or C terminus. I am asking this because on the basis of this we can see that whether the protein is expressing or not. if your protein is present at N term of the GFP than because of your protein's low expressiveness the GFP expression will also be affected.
try to run your construct H2B-GFP plasmid with GFP-plasmid (from any other lab) then scan the expression, you not need to western blot, if it correctly expressed, it will give you a strong signal.
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