From: ccarrawa@mednet.med.miami.edu
Date: Fri, 24 Jan 97 07:49:27 EST
Message-Id: <9700248541.AA854120911@mednet.med.miami.edu>
Subject: keratin
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Another major source: SDS PAGE electrophoresis buffer. At one
time while running IEF/SDS PAGE and SDS/SDS 2D gels, we were finding
massive amounts of bands in the 50-60 kDa range, all the way across our
second dimension gel. A systematic analysis of the problem (i.e.,
running samples of several potential culprit buffers on SDS PAGE)
showed that our SDS solutions were heavily contaminated. The solution
containing mercaptethanol was more heavily contaminated than that
without. In fact, our stock mercaptoethanol solution was also
contaminated. A "keratin" standard was prepared by dipping a finger
into a sample of each buffer, a VERY effective elution method!
Lesson for students of all ages: No experiment is cleaner than the
reagents used. Solutions: Aliquot out newly opened bottles of
stock reagents into small containers; make up small volumes of final
solutions; don't use anyone else's reagents and don't allow yours to
get into general circulation; DON'T TOUCH ANYTHING WITHOUT GLOVES.
The point made yesterday about contaminated glass- and plasticware is
also a VERY valid one.
Sunny Carraway
Miami
Weather inconsequential: We've had a FABULOUS winter here! Wish you
all could have spent a couple of weeks here around Christmas.
---------------------Reply separator--------------------------
One of the most common sources of keratin(?) contamination is buffer B (1M
tris, pH 6.7) used for the preparation of stacking gel of the standard
tris/glycine polyacrylamide gels. We routinely keep the buffer refrigerated
for no longer then 4 weeks - then discard it and prepare fresh. Our buffer B
contains 0.05% azide. If the buffer goes wrong then there is a double band
across the gel visible after silver staing of the gel or colloidal gold
(AuroDye) staining of the membrane. Seeing the same band after CB staining (or
amido black on the membrane) indicates severe contamination, usually not _only_
buffer B related. The molecular weight suggests keratin, but I have never
attempted to identify it by sequencing. Has anybody?
Jacek Mozdzanowski
Analytical Sciences
SmithKline Beecham
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From: Jacek Mozdzanowski-1 <Jacek_Mozdzanowski-1@SBPHRD.com>
Date: 23 Jan 97 12:00:03 EDT
Subject: keratin
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