I am looking at an NBD labeled lipid. I see a large enhancement of the excitation intensity but very little in the emission intensity upon addition of protein. I was wondering how this can occur and if anyone has seen this kind of behavior before. In general what does a large increase in the intensity at the excitation wavelength of a fluorophore mean?
There is no such thing as excitation intensity. There is only emission intensity. What you mean is that when you fix the emission wavelength setting and scan the excitation monochromator, you see an increased peak emission intensity when the protein is present compared with when it is absent. Perhaps what has happened is that the excitation and/or emission peak wavelengths have shifted due to the binding of the fluorophore to the protein (or to detergent micelles if the protein solution contains detergent). At the same time, the quantum yield may have increased. You should measure both excitation and emission spectra in the presence and absence of protein. In each case, the fixed monochromator should be set to the peak wavelength, if possible. At the end, you will report the peak excitation and emission wavelengths, and the relative intensities of the spectra. Ideally, you will also measure the quantum yield under each condition (i.e. +/- protein) by comparison to a standard substance with known quantum yield.
When you are recording the excitation spectra you are actually monitoring the emission at the emission maximum while exciting at different wavelengths. Therefore, you are actually getting the increased emission. If you cannot reproduce that in the emission spectra, there must be some technical problems. Check if you are using same slit width for excitation and emission measurements.
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