My protein has color of brown but in UV_Vis spectroscopy no distinct band after 280nm. At high conc. of protein I can observe at shoulder at 383nm so I excited my protein sample at 383nm and took emission spectra where I got the 450nm emission. I dont know what causing color to my protein and also dont know the interpretation of fluorescence spectra. Can any expert help me?
Work at lower protein concentrations to see if the emission peak shifts to shorter wavelengths. If it does shift to shorter wavelengths (i.e., to 348 nm), the fluorescence probably arises from some of the fluorescent amino acids (tryptophan, tyrosine, phenylalanine). Fluorescence reabsorption, which normally occurs at high fluorophore concentrations, can shift the emission peak to longer wavelengths. If the emission peak does not shift to shorter wavelengths upon reducing the protein concentration, the emission probably arises from some sort of contaminant. I think that the latter possibility is the most likely one. I've heard that some commercial buffers can contain fluorescent molecules. You could measure the emission spectrum of the buffer in the absence of protein to see if the buffer is the problem.
Work at lower protein concentrations to see if the emission peak shifts to shorter wavelengths. If it does shift to shorter wavelengths (i.e., to 348 nm), the fluorescence probably arises from some of the fluorescent amino acids (tryptophan, tyrosine, phenylalanine). Fluorescence reabsorption, which normally occurs at high fluorophore concentrations, can shift the emission peak to longer wavelengths. If the emission peak does not shift to shorter wavelengths upon reducing the protein concentration, the emission probably arises from some sort of contaminant. I think that the latter possibility is the most likely one. I've heard that some commercial buffers can contain fluorescent molecules. You could measure the emission spectrum of the buffer in the absence of protein to see if the buffer is the problem.
383 nm is too long a wavelength to be due to tryptophan. I think your protein is probably contaminated with colored material from the cells. Try running it through a gel filtration column or do some other purification step to remove small molecule contaminants.
I think there is some contamination in your protein sample.Generally color of protein solution is transparent. As far as your's question concern normally protein absorb at 280 nm (tryotophan absorb around 295) and emits at 340nm. now in ur case your protein absorb around 380nm that too far from normal range. so my suggestion to you first you confirm your protein sample having no contamination then you dialyzed your protein solution with working buffer.
Hope you get rid off from this problem.
Thanks
The protein sample was S-200 gel filtered and it has a nice mono-disperse peak at standard value. Also I've changed the buffer 3-4 times and color is still there. So I am confident that my protein sample is purest as it gets and I have took the buffer emission spectra and there is nothing that contributes to my protein. So again back to square one.
Cant do the native as acrylamide is one of the substrate but sds-page give clean bands after gel-purification.
If the colored component is not covalently attached to your protein (check by mass spec) and also does not separate from it by gel filtration, then it maybe an aggregated material with a size similar to that of the protein. You could try sucrose gradient centrifugation to separate them, assuming the substance has a different buoyant density from your protein. If your protein is stuck to it, though, this may not work. A little detergent may help separate them in that case.
???? My only Q is that it is colored but why is not giving the absorbance in UV-Vis spectroscopy. Protein activity is fine with the purification level (~99%). did the Fluorescence and no information. Even changed the buffer with inorganic/organic buffers. Problem with me is that I've set-up the crystallization of this protein and some hits have colors and some not. So if my protein is really colored then its crystal will be. Or if some other contaminants causing color then protein crystal will not be colored. So with what information I should proceed for diffraction stage and data collection.
A brown color indicates that absorbance is occurring over a broad range of wavelengths. Since there is no substantial peak in the spectrum, to me this suggests contamination with a heterogeneous substance. Since you have both uncolored and colored crystals, you may soon have your answer if any of them diffract.
Finally got the structure of protein and did the XFS and found that it has mononuclear Fe coordinated with Y,Y,G residues. Adam B Shapiro Sergio Gabriel Lopez Md. Fazle Alam
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