I have made a gfp fusion gene. When I saw the gfp signal under a fluorescence microscope there were much gfp background signal. The gfp gene follows my gene (promoter-my gene-gfp gene-terminator). I did not delete the start codon of gfp gene. There are three error bases in the fusion gene (error bases may have been made during PCR amplification). Can GFP start codon make background signal? I have attached a photograph that expresses the backgound signal.
What is the size of your gene?
What cells are you using?
Dead or dying HEK cells often give strong green fluorescence.
Look at the bright field images as well and see if the GFP+ cells look worse than the rest of the population. Also check untransfected cells, usually the regular GFP filter sets give you quite some background.
If you are really worried about alternative translation initiation do a Western blot against GFP and see if you get multiple bands.
If your gene of interest has a a large sequence, having a alternative translation initiation should be very rare. but the transfected cell and the construct and also the vector itself may affect this background noise. you should provide a little more data to have a better troubleshooting for that.
Generally it may not.You check by western by using antibody against GFP or protein of interest.
all the best
No - the start codon will not cause background fluorescence. It is not normal to remove the start codon as this encodes methionine which may or may not be required, only deletion of the prior stop codon is required. It is normal to include a flexible linker between the 2 genes so that your protein and GFP have opportunity to fold correctly. As others suggest, western blot is required to confirm successful and stable fusion of your 2 proteins, however this does not imply functionality of either protein (although assuming your image is GFP fluorescence rather than immuno, GFP certainly looks functional - this is too bright to be autofluorescence. The round cell is likely dead/dying. You would still need to test functionality of your protein.
Why do you think that this is background signal? If you have used transfection and a strong promoter, you are highly overexpressing your fusion protein. Functional or not, it is probably saturating its normal system and not all of it will be correctly localised. Thus, fused to GFP, this would give you the appearance of total-cell fluorescence.
Thank you everybody for many opinions. the opinions are very helpful for me.
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