Hi there,

Firstly, in answer to your question. The maximal absorbance for ATP is at 259 nm, but I would not have thought 10 mM would present you with serious problems when taking readings at 280 nm; at least, I certainly have encountered no problems.

I'm not sure I quite understand your problem though. Personally I would expect there to be a large absorbance at 280 nm if I was looking at purified protein fractions. Whilst the maximal absorbance wavelength should be determined empirically for each new protein, the standard practice, as I'm sure you're aware, is to use the absorbance at 280 nm, together with the predicted molar extinction coefficient to determine the protein concentration. If you are getting higher values than you think you should be seeing then perhaps your protein has become unfolded, as unfolded proteins frequently yield a greater absorbance due to the exposure of aromatic residues that would otherwise be buried in the native structure. Without knowing more about your situation, and the type of protein you are trying to purify, I could not offer much more specific advice.

Whilst I am a great advocate of MAD Scientist for answering many different types of questions on many scientific subjects, questions of this particular technical nature may be better suited to an online techniques forum where you will benefit from a more expedient answer, and from multiple opinions from many other research scientists. I would recommend a site such as the [NWFSC Molecular Biology Techniques Forums], which is a well respected, high quality technical forum.

Hope that helps,