Peptide concentration determination

[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 for any help!

[CharonY]

If you are certain that only the N-terminus is labelled, it should work. Other possibilities include amperometry, weighing (very tricky). Or one could hydrolyse them and perform AA anlysis (a bit tedious and would not work with mixtures, but that does not appear to be the case here).