Three caveats and a caution: The caution is that we used the values for a 1mg/ml protein solution above, which is a 0.1% protein solution. This is used by many authors, textbooks, lab manuals, but some others use a 1% protein solution, or 10mg/ml. You might have to look carefully to see whether a particular, paper, protocol or whatever uses absorbance values for 1% or 0.1% solutions- if the values appear to be about 10X of ours, they are most likely using absorbances at 1%. First caveat, Cystine, in other words two Cysteine residues linked by a disulfide bond, also have a small absorbance at 280nm, but the effect is small (110 units for a 1M solution), and to calculate how much this influences the absorbance you have to know how many of the Cysteine residues are disulfide linked, which you can't determine from the sequence alone. So we ignored the Cystine effect, since Cysteine is also a rare amino acid (~2% in the average protein), and only extracellular proteins have a significant number of disulfide links. Second caveat, contamination by substances with aromatic rings can mess up your calculations; each and every DNA and RNA base contains either one or two aromatic rings which all absorb strongly at 280nm, so contamination by even small amounts of nucleic acids can be a particular problem. Third caveat, the pathlength of your cuvette is assumed to be 1cm, which is standard in most spectrophotometers. If the pathlength is less than this, then the concentration measured should be corrected to take this into account.
Disclaimer: This
program was constructed primarily to save time for busy researchers
around the world. We hope you find it useful, and we are confident
that it is accurate and reliable. However, we cannot be held responsible
for any problems which may arise as a result of the use of this program.
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