Jennifer,
Another method of concentrating proteins from dilute samples prior to
SDS-PAGE is described in the following paper.
Ziegler,J et al. (1999) Analytical Biochemistry 250:257-260
Good Luck!
David Avila
Basel Institute for Immunology
-----Original Message-----
From: Jennifer Norton [mailto:jnorton@interchange.ubc.ca]
Sent: Wednesday, July 26, 2000 1:10 AM
To: Recipients of ABRF List
Subject: concentrating protein
Hello,
I have a client who wants me to N-terminal sequence a ~70 kDa protein.
Presently, he has a partially purified prep of approimately 3 mL (from 90
mL) in which he estimates that he has only 4-5 bands when run on a gel and
that he only has 0.01 ug of the band of interest (read: very tiny band)/20
uL when loaded and run on a gel.
He has tried to concentrate it using a centricon filter but found that he
lost a great deal of protein. Since it is not a pure prep I am assuming
that using a ProSorb cartridge won't be of any use. He asked me if he could
do a TCA precipitation and then resuspend in a smaller volume for loading.
I checked the ABRF archives but couldn't get specific protocol/application
info. Would you expect that to work? Any hints?
Another suggestion was to do an acetone precepitation, but when I checked
the archives I couldn't find any matches to my search. Would this be a good
pre-gel running method of concentrating his protein?
Thanks for your help,
Jennifer
Jennifer Norton, M.Sc.
Peptide/Protein Sequencing and Peptide Mapping
Nucleic Acid Protein Service (NAPS) Unit
Biotechnology Laboratory
University of British Columbia
Room 237 - Wesbrook Building
6174 University Boulevard
Vancouver, BC, Canada
V6T 1Z3
Tel: (604) 822-9688
Fax: (604) 822-0676
jnorton@interchange.ubc.ca
http:/www.biotech.ubc.ca/services/naps/
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