As far as I am concerned,there no simple nor accurate way to determine
peptide content.
I have been using amino acid analysis for years,and,yes,there are
drawbacks.Essentially,
you need to have someone weigh accurately your peptide,and everything else
is left to
the amino acid analysis.For the amino acid analysis,I examine all amino
acids,and,then
calculate the mean value.That value will give the nmoles of peptide
present.Typically,I
see that the content is 65-75% of the weighed sample.No special precautions
are taken to
get a constant weight of peptide.I just like to weigh as much peptide as
possible,dissolve
it,and take an aliquot for analysis.Direct weighing of dried samples tend
to give values
which off to a greater extent.If all pipettes are well calibrated,and your
pipette is really pure,the amino acid analysis can be accurate within +/-
5%.
"Alessandro Tossi" <tossi@univ.trieste.it>@aecom.yu.edu> on 05/29/2000
04:15:11 AM
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Subject: peptide quantification
Is there a way of quantifying a peptide which is both simple and accurate?
Weighing is often a problem due to crystallization waters and often the
available peptide is insufficient. We have tried calculating the global
peptide bond extinction coefficient, used the bicinconinic acid and
ninhydrin assays, used
the method of Waddel and others, and results always vary considerably, and
do not agree too well with estimates based on internal standards (Tyr or
Trp), when these are available. We have also tried leaving the Fmoc group
on
to provide the standard, and then removing it, but its messy and apparently
sometimes results in peptide degradation. Its a real problem. Amino-acid
analysis is claimed to be the most accurate method, but I have been told it
has problems as well. Can anyone suggest alternative methods?
Alex Tossi
*********************************
Dr. Alessandro Tossi
Department of Biochemistry
University of Trieste
Via Giorgieri 1
I-341227
ITALY
*********************************
email: tossi@bbcm.univ.trieste.it
phone - 39 040 6763673
fax - 39 040 6763673
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