From: bcdchin@muccmail.missouri.edu
Date: Thu, 23 Jan 97 16:55:49 CST
Message-Id: <9700248541.AA854115554@muccmail.missouri.edu>
Subject: Re: keratin
To: Recipients of ABRF List <abrf@aecom.yu.edu>
I have had severe contamination 7 years ago, moved and have not had the keratin
problem since. Where it came from, I'm not sure. Maybe mice and rats in the
lab at night, maybe skin flakes from the hands, hair or beard? It's just
easier to keep getting fresh reagents and always wear gloves (mandatory lab
rule, remember it only takes one ungloved finger to contaminate a whole
bottle). DDT has been reported as a sources of keratin containation, but I
don't know if that was introduced in the lab or at the mfg. site. I remember
that the old ultra pure chemical analysis (Smith?) listed "rat hair" as a
contamination.
I don't recall the reference but the keratin contamination was identified when
silver staining and see the band only when DTT was used. Later it was id as
keratin by what means? It also common with Western's (antibody staining).
David Chin
UMC Protein Core
--------------------------------------------
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