Dear all -
I wanted to thank everyone for their input on how to go about
determining levels of SeMet incorporation in recombinant proteins.
Here is the summary of suggestions:
1) MALDI-TOF:
http://www.ibs.fr/Plates-formes/Caracterisation-de-proteines/Spectrometrie-de-Masse/
http://www.ibbmc.u-psud.fr/spip.php?rubrique41
2) Amino acid analysis:
http://keck.med.yale.edu/prochem/aaa.htm
3) microPIXE:
http://www.ionbeamcentre.co.uk/index.php?target=3:68
We have successfully used microPIXE (see the link for an excellent
review of the method) in collaboration with Univ. of Surrey, and this
proved to be the most precise way of determining Se levels in our case
- we are dealing with a glycosylated protein. We tried mass spec, but
this didn't allow us to quantify incorporation well because of the
broad peaks obtained for the sample due to glycosylation.
Best wishes and thank you again,
Marija
On Sep 28, 2009, at 4:50 PM, Elspeth Garman wrote:
Dear Marija
This can be done by Proton Induced X-ray Emission on a tiny sample
(0.2microl) of liquid protein to get the sulphur to selenium ratio
and thus work out the substitution of Selenium.
See:
http://www.ionbeamcentre.co.uk/index.php?target=3:68
and contact
Dr. Chris Jeynes for this service:
c.jey...@surrey.ac.uk
Best wishes
Elspeth
Marija Backovic wrote:
Dear all -
Does anyone know of a facility that provides amino acid analysis
for determination of SeMet incorporation in a recombinant protein?
I contacted centers at Grenoble and Uppsala, but SeMet
determination is not something they routinely do.
Any suggestions would be highly appreciated!
Best,
Marija
Marija Backovic, Ph.D.
Pasteur Institute
Department of Virology
25 rue du Dr Roux
75015 Paris
France
+33 (0)1 45 68 82 87
--
******NOTE NEW E-MAIL ADDRESS AND PHONE NUMBER************
----------------------------------------------------
Elspeth F. Garman,
President, British Crystallographic Association
Professor of Molecular Biophysics, University of Oxford
Visiting Professor in Chemistry, University of Durham
Postal address:
Laboratory of Molecular Biophysics,
Department of Biochemistry,
University of Oxford, Tel: (44)-1865-613297
South Parks Road, FAX: (44)-1865-613201
OXFORD, OX1 3QU, U.K. E-mail: elspeth.gar...@bioch.ox.ac.uk
http://www.biop.ox.ac.uk/www/garman/gindex.html
-----------------------------------------------------
Marija Backovic, Ph.D.
Pasteur Institute
Department of Virology
25 rue du Dr Roux
75015 Paris
France
+33 (0)1 45 68 82 87
