I have a peptide dissolved in miliQ water with one tryptophan. How can i measure the concentration of my peptide from tryptophan fluorescence? Please give me in details, i am not in this field?
Thank you in advance.
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Posted 08 October 2013 - 11:02 AM
excitation wavelength 280nm, emission wavelength 348 (see this webpage). you can also look at this webpage.
unless you need to use fluorescence, you can read absorbance at 215nm (or a little lower). this reads peptide bonds.
Posted 08 October 2013 - 12:13 PM
thank you so much. But it will give only tryptophan fluorescence. How can I measure concentration of the peptide? do I need to calculate from any equation? do I need to do standard curve?
Posted 09 October 2013 - 03:29 AM
yes, you would use a standard curve (with absorbance you could use a specific extinction coefficient to calculate concentration, there may be a similar method with fluorescence but i'm not familiar with it).
optimally, a standard curve should be prepared with a purified sample of the peptide or protein of interest. it could be prepared with a similar peptide that has one tryptophan or another peptide with a known number of tryptophans (maybe with purified tryptophan?).
Posted 09 October 2013 - 09:33 AM
Thank you very much.
Do you know the formula with extinction coefficient ? Sorry to ask very silly stupid question, I know.
Posted 10 October 2013 - 03:51 AM
you divide the reading by the extinction coefficient and then apply other factors (eg dilution).
extinction coefficient will be M-1 cm-1 for absorbance (see this wikipedia page).