In order to proceed with the quantification of an Azide Alkyne Cycloaddition reaction efficiency by using a fluorogenic probe with Cy5-DBCO, I first need to construct a calibration curve. The problem is that water gives me fluorescence intensity values as well at an excitation wavelength of 647 nm and emission of 668 nm (excitation and emission that correspond to Cy5). I observe this using just water as a background control and it shows really high values. I am using a TECAN infinite 200 instrument and the samples are prepared in a black polystyrene 96-well plate.
Thanks for your reply, but it is not cross contamination. I repeated the blank (water) measurement independently and it continue showing the same value.
Well, no other reason, but cross contamination, is conceivable. Have you tried fresh wellplates, empty wellplates, without wellplates?
Hi Juan,
I think you have the M200Pro based on your ability to select wavelengths that are not rounded numbers, is that correct? This is a monochromator based fluorometer that has continuous control over wavelength, and the same system I have. If you have the F200, then you have filters that select wavelengths and I believe the same argument will apply, but the specifics will be a little different.
The M200Pro has a 9 nm bandpass on excitation and 20 nm bandpass on emission. Those numbers mean that the full width half maximum band of light that is passed by the excitation and emission monochromators (the things that choose wavelength) will be 9 nm wide and 20 nm wide respectively. At 647 and 668 nm, you have overlap between the two (see first attached image). This means you will be scattering excitation light into your detector. Black plates will help minimize this, but they won't eliminate it. Excitation light is much much more intense than emission light.
If you can shift either excitation or emission to widen the gap, the problem should go away (see second image where emission is shifted to 685 nm). Excitation and Fluorescence bands are generally pretty wide, so this should be a possibility. If you absolutely can't move the wavelengths any further away, then I recommend changing the z-height if you have that option on your instrument. This changes where the light is focused in the well and may reduce the scattering of light. I doubt it will remove everything though. Hope that helps!
I wasn't paying attention to your dye. Here are the spectra for Cy 5 to give you an idea what wavelengths you can use. The ones you chose are the most intense, but not well suited for your instrument.
https://www.chroma.com/spectra-viewer?fluorochromes=152
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