I am going to label a membrane protein which has very few expression and is focus localization with GFP. I has successfully fused GFP to the protein C-terminal in the genome DNA. But I don't see clear fluorescence when detected by confocal microscopy. Do anybody good ideas for this experiment? Thanks so much.
Hello, In cells where the gene is expressed, and the tagged proteins are produced, GFP is produced at the same time. Thus, only those cells in which the tagged gene is expressed, or the target proteins are produced, will fluoresce when observed under fluorescence microscopy. See attached info may be useful for you. Good luck!
If you do not get a good fluorescence signal for your fusion protein, there are several possible explanations:
- Your fusion protein is not expressed in sufficient amounts (you may need to confirm expression levels with alternative methods, e.g. western blot, to see whether the protein itself is present at a level where you should be able to detect it, and may need to tweak the expression system for higher expression)
- The fusion interferes with proper maturation of the GFP fluorophore (Protein aggregates before GFP can fold, or sterically hinders GFP folding. You may need to play around with the linker length and other aspects of the expression construct)
- Your protein is degraded too quickly
I would check multiple lines, at least 10 or so, because the fusion expression and stability may differ from line to line, depending on the transformation. Also, when I have had difficult to express transformations I have used a higher expressing, constitutive promoter and/or made constructs that also included N-terminal or internal fusions. Sometimes the location of the GFP affects stability or degradation. Best of luck.
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