attached mail follows:
Suzanne:
See Paul Jeno's paper in Anal. Biochem. 215 (1993) 292. He used Hydrophilic
Interaction Chromatography (HILIC) with an electroeluted membrane protein.
Coomassie Blue, salts and SDS eluted in or near the void volume. His protein
stuck quite nicely and was eluted with a gradient from 70-0% PrOH.
In your case, I would encourage you to try 85% ACN instead of 70% PrOH, and
use 10 mM ammonium acetate or formate (pH 3-4) instead of the 50% formic acid
in the mobile phases that Paul used. If you do this with an HPLC column
suitable for HILIC, you will get a nice resolution of your tryptic peptides
with no need for recourse to reversed-phase. However, if all you want is to
get rid of the Coomassie Blue on a small scale, then consider using a
SuproTip from Amika Corp. (www.amika.com) with some HILIC packing material
glued to the walls. Bind the peptides in 85% ACN with 10 mM ammonium acetate
or formate (pH 3-4), rinse the SuproTip a couple of times with the same
solvent to wash out the Coomassie Blue, and wash out the peptides with a
microliter or two of water containing the salt but no ACN.
Regards,
Andy Alpert
PolyLC Inc.
9151 Rumsey Road, ste. 180
Columbia, MD 21045 USA
tel: (410) 992-5400 FAX: (410) 730-8340
**************************************************
<< Subj: zip tips and coomassie- trypic digest
Date: 03/07/2000 6:23:34 PM Eastern Standard Time
From: perry@interchange.ubc.ca (Suzanne Perry-Riehm)
Sender: abrf-request@aecom.yu.edu (Association of Biomolecular Resource
Facilities)
To: abrf@aecom.yu.edu (Recipients of ABRF List)
Any thoughts on the use of Ziptips to possibly clean up a tryptic digest
of an electro-eluted sample from SDS PAGE? The sample was sent from Peru-
highly coloured with Coomassie and I now need to separate the resultant
peptides on my PE ABI173 cHPLC and am loath to inject such a mess.
Any thoughts/protocols on cleanup of Coomassie from a peptide containing
liquid would be greatly appreciated.
Regards,
Suzanne Perry-Riehm
Suzanne C. Perry, Research Associate
NAPS Protein Sequencing and Peptide Mapping Laboratory
Biotechnology Department, University of British Columbia
CANADA
phone:(604)822-9688( direct) or (604)822-9662 (main) Fax:(604)822-0676
email:perry@interchange.ubc.ca
Visit our web page at : www.biotech.ubc.ca/naps/napshome.htm
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