Sensitive peptide assay?
Marcus, Dr J.
J.Marcus at botany.uq.edu.au
Wed Nov 6 17:37:27 EST 1996
> I wonder whether there is a sensitive peptide assay being
> able to detect between 10 pmol and 1 nmol of a peptide. I
> was thinking about UV detection at 214 nm but I think that
> the extinction coefficient is not high enough (when I
> assume that it's 1000 per peptide bond -- which is just a
> wild guess -- I would be able to detect 1 nmol / 100 ul).
> Any ideas?
Do you need to know the concentration or just find out if
your peptide is there?
Assuming you want to quantitate, here are some thougths
that may or may not be useful...
1) A very good method for accurate quantitation is acid
hydrolysis followed by amino acid analysis.
2) Perhaps a less time consuming way would be to run your
peptide on an RP-HPLC column and look at 214nm absorbance.
100 microgram/ml concentration should give an absorbance of
around 1 at 214nm. Peaks of 0.01 Absorbance should easily
be detectable; that translates to 1 microgram/ml
concentration. If your protein peak is about 0.5 ml in
volume then you would have 0.5 micrograms of protein. So,
if your protein is say 10,000 Da you would be detecting 50
pmols of protein. If your protein was 100,000 Da your
would be detecting 5 pmol. Not bad if you ask me. The
nice thing here is that you can get your protein back.
3) If you have enough sample and your don't mind using some
of it up you can do a micro BCA assay. Pierce sell the
reagents but the mix is easy to make yourself.
I hope that is useful.
John Marcus Marcus at tpp.uq.edu.au (Dr J.Marcus)
Cooperative Research Centre for Tropical Plant Pathology
5th Level John Hines Building
University of Queensland
St. Lucia, QLD 4072
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