Date: Fri, 24 Jan 1997 10:03:39 -0500
Message-Id: <v02130514af0e337e9dff@[128.231.120.108]>
From: lmarekov@Box-l.nih.gov (Lyuben Marekov)
Subject: Re: keratin
To: Recipients of ABRF List <abrf@aecom.yu.edu>
>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
As far as I recall the 2 bands of MW 54K and 68K were
attributed to the presence of (oxidized) mercaptoethanol in
the sample buffer - see Practical Protein Chemistry
(1986), p302 ed. Darbre, John Wiley & Sons and reference therein: Tasheva &
Dessev, Anal Biochem (1983) 129, p98.
But this does not completely exclude keratins - they are
in the air as John W Crabb wrote:
"...Many of us see human keratin sequences from time to time, particularly from
electrophoretically purified samples. My best guess is that it comes from
dandruff and probably introduced into one or more of the electrophoresis
reagents during manufacturing and/or packaging...".A more
likely source is I believe also especially if someone in the lab has dandruff!!
Lyuben N. Marekov
NIH, NIAMS
Bldg 6/Room 128
Bethesda, MD 20892-2752
phone (301)-496-7190
fax (301)-402-2886