[jnh33]

Hi everyone,
 
I have some 20-aa peptides which I've had synthesized. The problem is I need to determine their concentration after they have been dissolved in water. (They don't dissolve fully so it's not possible to simply consider the weight of the lyophilized peptides beforehand).
 
Each peptide has a fluorescein molecule attached at the N-terminus. So one way I've been doing it is to measure the absorption of the peptide solution at 490 nm (the absorption of fluorescein). I can then compare this absorption with absorption values of known amounts of free fluorescein at the same wavelength to calculate a molar concentration of the peptides. Do people agree with this method? Are their reasons why it might give wrong readings?
 
Are their other methods you have used or can think of? I read about BCA assays but the minimum size of peptides required is 3kD which is bigger than what I have. Also taking the absorption at 280nm doesn't work well because my peptides don't contain the Trp, Try, and cysteines.
 
Thanks!

[Babcock_Hall]

I don't see any obvious reason why your technique won't work.  Amino acid analysis is another option, although the presence of a derivative with fluorescein might make this difficult.  Could you dissolve your peptides in an organic solvent or mixed solvent, to lessen the precipitation problem?

[Yggdrasil]

Quantifying concentration based on the absorbance of fluorescein seems like a good idea.  Sometimes the extinction coefficient of fluorescent dyes are sensitive to environment (for example, fluorescein is fairly pH sensitive), so there are some reasons to think the extinction coefficient of the free dye may differ slightly from that of the protein conjugate, but I wouldn't worry too much unless you need highly accurate values.

Alternatively, peptide bonds absorb at 205 nm and this absorption can be used to quantify the concentration of small peptides.  See Scopes Anal Biochem 59: 277 (1974) http://www.sciencedirect.com/science/article/pii/0003269774900347