Measuring peptide concentration

JB j.bukczynskiDELETE_ME at utoronto.ca
Tue Mar 19 01:09:54 EST 2002


Thanks guys. Much appreciated...

Jacob

>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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