I have tagged my protein of interest with mCherry at N terminus and have episomally expressed in Leishmania promastigotes. I observed even in live cell fluorescence stars quenching within few seconds. Though mCherry is known for its resistance to quenching. Can any body suggest what short of problem I am facing. Earlier I tagged my protein at C terminus with Gfp. Latter found that gfp is not fusing with my protein, so I used mCherry at N terminus.
May be the concentration is high. so check once by diluting the solutions, probably it should work through. These fluorescent molecules tend to interact with each other at higher concentration and results in quenching.
Perhaps the illumination is too bright, causing photobleaching. Here is an article that compares photobleaching rates for fluorescent proteins.
http://www.tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Methods%20-%20Choosing%20fluorescent%20proteins.pdf
I agree with Dr Shapiro: The excitation light source is too strong. You should try to reduce the intensity of excitation.
In addition, you'd better give up to detect the light by your naked eyes, and use CCD or CMOS cameras instead (they are much more sensitive than human eyes).
And if available, open the excitation shutter only when necessary.
- Badges
- Science topic
- Similar topics
- Chemistry
- General Biochemistry
- Proteomics