Hank-
I can think of three easy ways to quantitate your protein:
1. Reaction of the amine with fluorescamine, and analyze by fluorescence. By coincidence, there was a communication by Rodionov on this topic in today's Roundtable communications, in which he provided references. 2. Disulfide bonds do absorb weakly at 280 nm. If you have enough material, you could determine the peptide concentration by UV at this wavelength. There are published molar extinction coefficients for disulfide bonds, or you could generate your own extinction coefficient for the peptide.
3. HPLC analysis. Set up a standard curve with peak area (215 nm) vs amount injected.
Vernon
Vernon A. Shoup
Regeneron Pharmaceuticals
Rensselaer, NY
(518)488-6012
vernon.shoup@regpha.com
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I am looking for a sensitive, linear, quantitative metod for determining
peptide content. The restrictions I am under are the following: I have a
12-residue peptide with one primary amine, three disulfides and no
aromatic amino acids. Also, I do not own an amino acid analyzer or
protein sequencer, but have good UV, fluorescence, CE and HPLC
equipment. Any suggestions? References to journal articles would be an
added bonus.
Thanks,
Hank Wolfe
henry.wolfe@nycomed-Amersham.com