Re: PHOSPHOPEPTIDE SEQUENCING
Laurey Steinke (lsteinke@molbio.unmc.edu)
Tue, 3 Mar 1998 10:12:55 -0600 (CST)
Dear Robert,
We have been identifying sites of phosphorylation for several customers
using cycles sent to us by Richard Cook of Baylor. This involves changing
S3 on the sequencer to MEOH/H2O and labelling the protein with P32 before
in-gel digestion. Peptides are then attatched covalently to a sequelon
membrane. We tried the technique with PVDF to see if we could get any
data, however the washout was horrible.
Your other option is LC/MS after in-gel digestion, but I have no
experience with this technique. If you are interested in the details of
the cycles I can get them to you. We have dedicated our old 477 to
phosphopeptide sequencing, and have better luck than trying to switch back
and forth. Ken Mitchelhill has been getting good results on an HP and has
a paper out on the techniques.
-laurey
Weather Inconsequential: A gray, moderately cold day. Walked in to work
anyway, for once I don't have an appointment in the middle of the day or
string instruments to schlep around.
Laurey Steinke
Protein Structure Core Facility
University of Nebraska Medical Center
Omaha Nebraska, 68198-4525
Phone (402) 559-6647
FAX (402) 559-6650
lsteinke@molbio.unmc.edu