Why not carry out the reductive alkylation with the protein bound to the
PVDF? Just wet the PVDF with ethyl acetate (or methanol), immediately
transfer the PVDF to the reduction solution containing 6 M guanidine plus an
appropriate buffer, react, (optional rinse), transfer the PVDF to the
alkylating solution also containing 6 M guanidine, react and then wash the
PVDF with water. With the guanidine present, losses will be minimal (<10%
with ethyl acetate wetting). Solid-phase reductive alkylation is so much
easier (and cleaner) than solution-phase reductive alkylation. Also, you
might want to try alkylating with acrylamide in this format. It doesn't stink
like the VP's do.
Steve
====================
Stephen Tindall
Argo BioAnalytica, Inc.
Phone: 1-973-605-2100
Fax: 1-973-605-2104
StvTindall@aol.com
====================
Subj: Prot Seq: protein recovery from pvdf for 4-VP treatment
Date: 1/21/99 10:47:48 AM Eastern Standard Time
From: Thomas.J.Miller@usa.dupont.com (Thomas J Miller)
Sender: abrf-request@aecom.yu.edu (Association of Biomolecular Resource
Facilities)
To: abrf@aecom.yu.edu (Recipients of ABRF List)
Hello. I have a protein band on ProBlott pvdf which I would like to
recover and then treat with 4-vinyl pyridine for Cys characterization.
Are there any reasons why using the published protocols for protein
extraction from pvdf would interfere with the 4-VP treatment?
Thank you in advance for your comments. Regards, Tom.
Thomas Miller
DuPont Company
Protein Sequencing Lab
email: Thomas.J.Miller@usa.dupont.com
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