The method is that by Edelhoch, mentioned a couple of times already in this discussion.
You recommended "determining extinction coefficients experimentally". How is plugging number of specific residues into a formula constitute experimental determination?
It's also described in the paper by Pace et al., the same paper that the formula in ProtParam is from (ProtParam does not use the values determined by Gill & von Hippel). Last time I looked into this, the consensus was that the Edelhoch method is the most accurate method for protein concentration determination; more accurate than dry-weighing plus N-terminal sequencing, etc.
Nothing beats quantitative amino acid determination but it's one technique that requires specialized lab and good standartization. By now, it's almost a lost art.
I agree with Vaheh that absolute protein concentration is not critical in crystallization but let's not forget that the protein concentration enters, one way or another, into measurement of virtually every biochemical constant. 50-100% errors there can be extremely important.
- Dima
