I would like to quench the fluorescence of Alexa dyes to decrease their fluorescence lifetime. Therefore, I am looking for a compound dynamically quenching the fluorescence of these dyes. I have tried KI (potassium iodide) which worked for Alexa488 perfectly, weakly for Alexa546, and not at all for Alexa647. I have read on the ThermoFisher web site that QSY, DABCO and N-propyl gallate should work. QSY is only available as an amine- or thiol-reactive compound, and I would like to add it into the solution. I have never tried DABCO and N-propyl gallate. Before testing the whole Sigma catalog I would like to know if such a dynamic quencher is available.
Thanks for any insight,
Peter
Hi,
It has been reported that some aminoacids like Tryptophan (Trp), Tyrosine (Tyr), Histidine (His) and Methionine (Met) are suitable for Alexa-dye quenching through a combination of static and collisional quenching. In think Trp is the best option for Alexa647.
Best,
In the meantime I read a couple of papers. In this one:
http://dx.doi.org/10.1021/bc0341324
it is spelled out that "The quenching of carbocyanine dyes, such as Cy5, and Alexa 647 by amino acids can be almost neglected." How large concentration of Trp do you use to quench Alexa647?
Regarding your question about KI concentration 750 mM KI didn't do practically anything with A647 fluorescence, while 50 mM Ki quenched 50% of A488. We measured intensity, so I cannot comment on the type of quenching.
Peter
Don't forget that the lifetime of Alexa647 is only about 1 ns, hence one would have to go to relatively high KI concentrations to see any effect.
I have worked with the QSY quencher before, but in a setup where it was attached on the same polypeptide chain as the dyes. Unlike Trp or KI, which quench by a collisional mechanism (Trp also by static complex formation), QSY quenches through FRET, so no direct contact is required (and the chemistry of the dyes should be of less importance). Like any FRET mechanism, it will reduce the lifetime of the respective donor dyes.
I am however not sure how well it will quench Alexa 647 fluorescence, you need to check the overlap of the QSY excitation and 647 emission spectrum for that. Also, I have no experience with FRET between "unconnected" groups free in solution - I would assume you need millimolar concentrations of quencher for that, which might make the experiment quite expensive.
If you worry about the amine-reactivity/thiol of the QSY compound - those can be easily removed, in the case of the amine even by just letting it stand around for an hour in a slightly basic aqueous solution, which leads to the hydrolysis of the NHS.
Another option would be the BHQ-2 quencher, also available as carboxylic acid, which seems to quench fluorescence between 560-670 nm efficiently, also via a FRET mechanism.
If you are looking for a collisional quencher, I would also try metal ions, like Zn, Cu, Fe, Mn or Ni, also in mM concentrations.
Good luck!
I think you should use dark quenchers (black hole quenchers) which is based on FRET and do not emit any fluorescence. It is specific for certain wavelength and is generally used for qPCR experiments as well...
http://www.atdbio.com/content/35/FRET-fluorescence-quenchers
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