I am using an ALEX system to filter direct excitation and leakage of donor dye in red channel and it is possible to select the FRET populations by filtering. But still I have some bursts in the low FRET area. Do you know how to be sure about this area?
Dear Naghmeh, if you are doing ALEX FRET you can probably distinguish between a donor only population and a real low FRET state. Actually, if you sort your data by a stoichiometry criteria all donor only bursts have to be cancelled. Than your FRET histogram contains only real low FRET states.
Hope everything goes well, wish you all the best. Greatz ;-)
Nice to meet you here Richard,
Actually, i used a little bit higher concentration of sample to get enough bursts. So the donor only population comes down to 0.7 in stochiometry. Now, it is difficult to judge about it.
Another approach would be to use a control system that has both dyes on your molecule but have the distance between them sufficiently large so that you expect zero FRET. Then, perform the ALEX experiments and generate a FRET distribution for the molecules that have both dyes. You can then compare this FRET distribution with the FRET distribution from experiment that you think might have a low FRET population. An fast way to compare the two groups would be to normalize each distribution and then subtract them. If the difference is random, then they are likely similar.
Hi Naghmeh,
If you increase concentrations so that every once in a while you get multiple occupancy (D-only and A-only coincident bursts) in the observation volume it will show up as a dual-labelled low FRET burst. You have to work at VERY low occupancy and do ALEX and then and only then can you eliminate the artifactual "low-FRET" populations. Ultimately if your dual-labeling efficiency is not 30% or better you will end up having to push the limits of the concentration so much that you will invariable get some co-incident bursts or else have to wait for a long time during acquisition to get enough detection events. I hope this helps.
Hi Kambiz,
I agree with you, i reduced the concentration and laser intensity for both dyes. so, i totally removed the zero pick population. thanks for your help.
Dear Kambiz, I totally agree...
Dear Naghmeh,
I thought a little bit on your problem. Sometimes one need higher sample concentrations in FRET just due to the fact of affinity or low K_(what-so-ever)-values. Then you can go down to lower excitation/detection volumina. Look in the literature, their are several hints. One possibility is to use oil immersion objectives instead of water immersion. Another one is of course STED microscopy in FRET ... unfortunately a challenging task ;-) Third one is TIRF-FRET and so on ...
Dear Naghmeh,
You're quite welcome. Good luck with experiments.
Dear Richard,
I agree with you as well. If binding is the assay then you can only get around the Kd problem by working at high enough concentrations (I wish I had a PacBio ZMV system to solve all such problems!). In addition, FCS can also be a nice alternative if you want to work in the low nM range instead of the pM range.
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