From: lsiconolfi-baez@netmail.hscbklyn.edu
Date: Thu, 02 Jan 97 14:38:26 EST
Message-Id: <9700028522.AA852244306@netmail.hscbklyn.edu>
Subject: ProtSeq:bulk matrix prep
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Happy New Year to All,
I am not under time or volume constraints in my lab that Lisa is,
so I sequence peptides on Biobrene prepped glass fiber filters,
(having the 4 cartridge Procise is also a joy) but I have a few times
used methanolic biobrene coated PVDF. There is no need to Precycle
these. My limited use of this matrix allows neither a recommendation
or caution, but ABI data shows improved sequencing results on a series
of peptides prepared in ProSorb cartridges to which 100 ug Biobrene
was added. The Biobrene solution can be prepared as suggested in the
ProSorb Protocol (20 uL Biobrene, 10 uL 0.1% TFA, 70 uL methanol). 5
uL of this solution provides the 100 ug.
The method I used is not "in the books" but is a modification of a
procedure DOT-BLOTTING ONTO PROBLOTT that ABI sent me. I prepared 1 mL
of the methanolic biobrene, and dipped PVDF pieces into it. (This of
course loads an undetermined and undeterminable amount of Biobrene on
the membrane.) A little thought was needed to accomplish this coating
and air drying in a clean environment. The dried coated pieces were
stored, refrigerated, in an epindorf, in a dessicator, for later use.
Samples are applied in at least 40% acetonitrile.
Just brain storming, I can imagine that a whole sheet of PVDF could
be coated using pipette dispensed solution, if it was clipped on a
line suspended in an old fashioned glass liquid chromatography tank.
Greetings from N.Y.
Linda
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Linda Siconolfi-Baez
Core Protein Sequencing Center
SUNY Health Science Cntr.
Box 8
450 Clarkson Ave.
Brooklyn, N.Y. 11203
718-270-1267 (phone)
718-270-3316 (fax)
lsiconolfi-baez@netmail.hscbklyn.edu
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