Deb McMillen
Institute of Molecular Biology
University of ORegon
>From: Russell P Pickford @ SB_PHARM_RD
>Date: 06-Mar-98 08:42:55 AM
>Subject: AAA - Keratin Contamination
>Categories:
>
>Hi All,
>
> I am currently developing a protocol for amino acid analysis from PVDF
>blots. However, I am finding human keratin, and collagen to be a major
>contamination problem, despite the use of gloves etc.
> Does anyone else have any experience of this? My ideal solution would
>be some form of wash which would remove the surface contaminants, whilst
>leaving the electroblotted protein untouched. This would be performed
>before hydrolysis. As I do not prepare my own samples, a change in the
>blotting techniques would not really be practical, so I'm looking for
>damage limitation!
>
>
> Thanks in Advance
>
> Russell
Date: Thu, 23 Jan 1997 10:26:34 -0500
Old-To: abrf@aecom.yu.edu
From: fernaj@rockvax.rockefeller.edu (Joe Fernandez)
Subject: Re: keratin
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Sender: Association of Biomolecular Resource Facilities
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Steve,
Yes, we have done something like this to prove a point. The whole
lab passed arround a blank piece of PVDF with their hands, digested it and
got some nice protein sequence data that matches keratin.
To regulate this and monitor keritan contamination we have come up
with the following stategy
1) Assume keratin comes from everywhere (stock solutions for digestion,
preparation surfaces, gel preparation soutions and apparatus, etc.).
2) Replace all digestion related stock solutions weekly! Sometimes bottle
caps can be a continual source of keratin. Also sample tubes can be
contaminated if they are stored in an area that is used for the general
lab. We keep a separate stock of tubes that are only used for digestion.
3) Perform blanks as follows
A) exact digestion blank PVDF as with sample
B) solution digestion with no PVDF with enzyme
C) solution digestion with no PVDF or enzyme.
If there is keratin in any solutions or from sample handling there
will be more peaks present in blank B than blank C. There should be only a
few peaks if any associated with the enzyme.
D) keep track of blanks B and C over time to note any change.
4) Last but most important is to wear gloves at all times including
solution preps as well as sample handling.
With all of these safty precautions I can definitely explain to an
investigator that the keratin most likely came from their end and I usually
sit down with them to try and find where it came from. Hopefully it came
from handling the blot or gel prep as appears to be your investigator's
case rather then they have managed to purify keratin (sometimes a
possibility).
Hope this has helped.
Joe Fernandez
Rockefeller University
From: ccarrawa@mednet.med.miami.edu
Date: Fri, 24 Jan 97 07:49:27 EST
Old-To: abrf@aecom.yu.edu
Subject: keratin
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Sender: Association of Biomolecular Resource Facilities
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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
From: lmarekov@Box-l.nih.gov (Lyuben Marekov)
Subject: Re: keratin
Cc: abrf@aecom.yu.edu
To: Recipients of ABRF List <abrf@aecom.yu.edu>
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>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