Vernon,
One way to minimize air oxidation is to do sample manipulations under a
blanket of argon. When you vent the vacuum centrifuge, vent with argon.
During digestion blanket the reaction with argon. Also make sure all
buffers (HPLC, digestion, recon. etc.) are thoroughly degassed.
Hope this helps.
Mike
--------------------------------------------
Michael Curtis
Department of Cell and Developmental Biology
Upstate Medical University
750 East Adams Street
Syracuse, New York 13210-1834
Phone: 315.464.8511
Fax: 315.464.8535
E-Mail: curtism@mail.upstate.edu
--------------------------------------------
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>From: VERNON SHOUP <vernon.shoup@regpha.com>
>To: Recipients of ABRF List <abrf@aecom.yu.edu>
>Subject: Methionine oxidation, again
>Date: Thu, Mar 1, 2001, 7:06 AM
>
> Thanks to all who responded to my question about how to get rid of H2O2
> from protein solutions, in such a way that the protein would not be further
> modified. In summary, the suggestions were:
>
> 1. Gel filtration, either with prepoured columns or by FPLC.
> 2. Repeated microcentrifugal ultrafiltration (e.g. Nanosep or Microcon)
> 3. Addition of an excess of methionine.
> 4. Addition of catalase (References: N. Neumann, Methods in Enzymology,
> Volume XI, pp 485-487 (1967); FEBS Letters 455 247 (1999))
>
> DTT would probably work, but there was some uncertainty about whether or
> not DTT would reduce methionine oxide in the protein. Any comments about
this?
>
> Now for my next question. After we isolate protein variants by rpHPLC (C4
> or C18, Acetonitrile gradients, 0.1% TFA), we dry the solutions down by
> vacuum centrifugation, and then digest with trypsin for peptide maps.
> Sometimes we see almost complete oxidation of the methionine-containing
> peptides, but the extent of oxidition is erratic. How does one minimize
> this apparent air oxidation?
>
> Vernon
>
> Vernon A. Shoup
> Regeneron Pharmaceuticals
> Rensselaer, NY 12144
>
> (518)488-6012
> (518)488-6030 FAX
>
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