I purified extensively my membrane protein, ionic exchange, superdex 200, his-bind, etc. SDSPAGE, Bradford test, Western have all shown pure protein protein. This protein is not known to associate with nucleic acids, so why is the absorbance of this protein at 258nm? And no phenylalanine is not abundant, I have 9 tryp!
Dear Joe
The ratio of absorptions at 260 nm vs 280 nm is commonly used to assess DNA contamination of protein solutions, since proteins (in particular, the aromatic amino acids) absorb light at 280 nm. The reverse, however, is not true — it takes a relatively large amount of protein contamination to significantly affect the 260:280 ratio in a nucleic acid solution.
In 100% protein and 0% DNA this ration is 0.57 that mean protein also absorb 260 nm but in low level.
On the other hand, certain metals bound to proteins can give strong signals at wavelengths less than 280 nm. Maybe you have metals in the solution which are bound to the protein?
You can check metal presentation by several methods.
If you have any question or need other help please dont hesitate to contact me
Cheers
Tryptophan is the major contributor to the absorption spectrum (lamda max @ 280 nm) of proteins in the near UV, and tyrosine lesser so (lamda max @ 276 nm). The most useful trp absorption for proteins arise from from what is referred to as the 1La and 1Lb absorption bands - the lowest energy absorption bands. Their are also higher energy absorption bands which will contribute to the absorption at 260 nm. So I would expect the absorption at 260nm for most any protein not to be zero.
You might want to look up the following reference:
Sweeney and Asher, J. Phys. Chem., 1990, 94, 4784-4791.
Two possible sources of excess absorbance at wavelengths shorter than 280 nm are (1) the detergent, as mentioned by Sven Schenk, especially if it has an aromatic group such as Triton X-100, and (2) Rayleigh light scattering from aggregated material or detergent micelles. Rayleigh light scattering increases to the 4th power with decreasing wavelength and can therefore shift the peak absorbance to a shorter wavelength.
To test for Rayleigh scattering you should perform dynamic light scattering experiments. Apart from information about the presence of aggregate, it will give you information on the homogeneity of you sample which can be useful at a later stage.
Is there a distinct peak at 260 or are you simply seeing the shoulder of the 280 peak?
Assuming the answer to the first question is yes, and that your absorption is not related to the buffer composition (easiest way to test is obviously scanning a blank sample with just your buffer), and that there is in fact some sort of contamination: you can try the following:
The fastest and most obvious way to test for DNA contamination is to simply run a small aliquot of your sample on a DNA gel (and stain with a DNA specific dye, e.g. EtBR if you have it).
Alternatively you could also run a mass spec on the sample and check for spurious MW peaks.
Hi,
A very simple question: are you definitely sure that your protein doesn't bind GTP and or ATP at any stage?
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