Dear colleagues,
I constructed a protein expression vector. After transfection, my target protein and linked GFP was well expressed. However, the protein was dispersed everywhere in the cells, making quantitative imaging difficult. I would like to control the target protein to a low amount in cells. Do you know if there are any methods to reduce the protein expression in cells? For example, adding some inhibitor at certain time?
Thank you,
Ling
If the GFP fusion protein does not contain a retention signal then it will be dispersed in the cytoplasm. Now if excessive fluorescence is an issue then you can express it under the control of a weak promoter and/or go for stable transfection. After selection, surviving cells would contain one or two copies of the plasmid (and your fusion gene) and will not fluoresce as strongly.
The reduction of protein expression through external factors it may be add analogous materials of that protein or do not give proper stimulus for expression of protein
You could try using an inducible promoter as this would give you tighter control instead of constitutive expression which floods the cells i.e. Tet-On from clontech.
bw
Ian
We would use the EF-1alpha promoter to give a more modest level of constitutive expression compared with the CMV IE promoter/enhancer if very high level protein is not desired. The SV40 promoter is also a good one to try (such as the one that drives neo in pcDNA3 vectors). I could see detectable EGFP expression from the SV40 promoter but is was noticeably weaker than using the strongest promoters.
Good luck!
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