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How to measure concentration of a peptide by tryptophan fluorescence?


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6 replies to this topic

#1 farhima

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Posted 08 October 2013 - 09:23 AM

Hi all,
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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#2 mdfenko

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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.


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#3 farhima

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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? 



#4 mdfenko

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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?).


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#5 farhima

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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. 

Thank you 



#6 mdfenko

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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).


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#7 farhima

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Posted 10 October 2013 - 08:33 AM

Thank you very much, I solved the problem.






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