Hi,
I wanted to study the conformation changes in BSA using Three dimensional fluorescence spectroscopic analysis in a spectrofluorometer. I am using BSA (10 uM) for this analysis and my parameters for scan are emission wavelength scanning range is from 200 to 600 nm and the excitation wavelength scan range is recorded from 200 to 400 nm at 5 nm increments.
I am not getting the 4 characteristic peaks that are Peak 1, the Rayleigh scattering peak, Peaks 2 and 3 which are the two typical fluorescence peaks of BSA and peak 4 which is the second order scattering peak. Instead I always end up getting multiple peaks of equal heights at random wavelengths. What could be the reason for this? I feel my protocol may not be correct or some scan settings need to be changed.
Thanks in advance :)
You may be seeing water Raman scattering peaks, although they will not be at random wavelengths. They will be at a fixed offset from the excitation wavelength (about 3500 cm-1) when measured in wavenumbers, not in nm. As a result, the offset in nm changes with the excitation wavelength.
You should not allow the the emission and excitation wavelengths to be the same, as this allows a lot of light to enter the detector, potentially damaging it. The emission wavelength should always be longer than the excitation wavelength.
Tryptophan excitation maximum is at about 280 nm. The emission maximum is between 330 and 355 nm, and the emission peak wavelength does not change with the excitation wavelength (the intensity does). Therefore, it should only require 2 scans over a narrow range of wavelengths to capture the pertinent information: one excitation scan at a fixed emission wavelength, and one emission scan at a fixed excitation wavelength. The approach you are using seems unnecessarily elaborate. Also, there is no useful information about the protein in the Raman scattering peak or the 2nd harmonic peak, so there is no point in scanning over such a broad range.
You may be seeing water Raman scattering peaks, although they will not be at random wavelengths. They will be at a fixed offset from the excitation wavelength (about 3500 cm-1) when measured in wavenumbers, not in nm. As a result, the offset in nm changes with the excitation wavelength.
You should not allow the the emission and excitation wavelengths to be the same, as this allows a lot of light to enter the detector, potentially damaging it. The emission wavelength should always be longer than the excitation wavelength.
Tryptophan excitation maximum is at about 280 nm. The emission maximum is between 330 and 355 nm, and the emission peak wavelength does not change with the excitation wavelength (the intensity does). Therefore, it should only require 2 scans over a narrow range of wavelengths to capture the pertinent information: one excitation scan at a fixed emission wavelength, and one emission scan at a fixed excitation wavelength. The approach you are using seems unnecessarily elaborate. Also, there is no useful information about the protein in the Raman scattering peak or the 2nd harmonic peak, so there is no point in scanning over such a broad range.
In addition to the above I would recommend having a look at water on it's own. The following is an example of Rayleigh and Raman lines.
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