I am running an enzyme assay that measures the fluorescence of a cleaved substrate at 360nm ex and 445em. I also have a compound in the reaction that can be fluorescent if excited at 287nm. Will that interfere with the fluorescence signal of my substrate?
Fluorescence excitation and emission spectra of organic molecules are fairly broad. If the substance can be excited at 287 nm, it may also have some lesser degree of excitation at 360 nm and possibly some detectable emission at 445 nm. The amount of fluorescence from this substance that you can detect will increase in direct proportion to its concentration. It may be too small to measure, but if it is detectable, at least it should be a constant. As long as it is small, it should not be a problem. Measure it and see.
If the two components can be assumed chemically/electronically independent, at least with a good approximation, then no interference is to be expected. Increasing the excitation wavelength means decreasing the corresponding photon energy. Therefore when you are exciting the sample with a 360nm photon, the same has not enaugh energy to excite that component that need a 287 nm photon to be excited. The basic concept is that a photon is absorbed as a whole, it can not deliver a fraction of its energy while interacting with any other system
Fluorescence excitation and emission spectra of organic molecules are fairly broad. If the substance can be excited at 287 nm, it may also have some lesser degree of excitation at 360 nm and possibly some detectable emission at 445 nm. The amount of fluorescence from this substance that you can detect will increase in direct proportion to its concentration. It may be too small to measure, but if it is detectable, at least it should be a constant. As long as it is small, it should not be a problem. Measure it and see.
I must disagree with the previous comment. If you want to subtract the background fluorescence from the substance with 287 nm excitation from the signal in your assay, you should do so by measuring its fluorescence under exactly the same conditions as are used in the assay, including excitation at 360nm, except for the absence of the substrate. If it turns out that the fluorescence intensity of the 287 nm substance when measured under assay conditions is negligibly small, then you can ignore it. If it is substantial (i.e. similar in size to the fluorescence measured from the conversion of substrate to product) but constant, you may also be able to ignore it. If it is substantial and changes during the course of the assay (such as by photobleaching), you will have to measure its time course and subtract it from the full assay time course. If it is much larger than the signal from the assay reaction, then any attempt at subtracting it will be inaccurate, and you should rethink the assay design.
First see the absorption profiles of both. Then try to excite towards red edge of 360 nm substrate where 287 nm absorption vanishes. If profiles are available issue can be resolved by exciting at different wavelengths.
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