Measuring peptide concentration

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Mon Mar 18 21:43:50 EST 2002


I disagree on one point : Expasy server does NOT calculate extinciton if you
don't have certain types of AA's in your sequence.
For example 'AAAAAA' results in

-----------
Extinction coefficients:

As there are no Trp, Tyr or Cys in the region considered, your protein
should not be visible by UV spectrophotometry.
-----------

There's a reason for it - while some absorption at 280 will indeed be
present, it won't be characteristic, and it would be overwhelmed by
non-specific absorption of crud.

Otherwise, way to go Emir :)

A.G.E.
"Emir Khatipov" <khatipovNO at NOuchicago.edu> wrote in message
news:pyul8.311$s4.23478 at news.uchicago.edu...
> Use Protparam tool at www.expasy.ch/tools to calculate molar extinction
> coefficient of your peptide and use it for your spectrophotometric
> measurements. Peptides absorb light at 280nm even if they don't contain
any
> aromatic residues. I hope it is needless to say that you should use same
10%
> DMSO in PBS as a reference. If you believe there are impurities in your
> samples, I would suggest determining absorption spectra of your peptide
> solutions at ~200-350nm (or at least 250-330nm). If your sample is pure,
you
> should see a nice peak at around 276-280nm. Be aware that peptide bond
> absorbs at 214nm, but there might be a lot of interference from impurities
> you may have in your sample.
>
> Emir
>
>
> "JB" <j.bukczynskiDELETE_ME at utoronto.ca> wrote in message
> news:b87a9ugslasbigp8lm4dhkqc4p0soe0se7 at 4ax.com...
> > Hi,
> >
> > Does anyone know how to measure peptide concentration using a
> > spectrophotometer? I have it dissolved in 10% DMSO in PBS. Any help
> > would be appreciated. Thanks,
> >
> > Jacob
>
>





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