Message-Id: <v01530500af0d2dbe27f4@[129.85.13.54]>
Date: Thu, 23 Jan 1997 10:26:34 -0500
From: fernaj@rockvax.rockefeller.edu (Joe Fernandez)
Subject: Re: keratin
To: Recipients of ABRF List <abrf@aecom.yu.edu>
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
>I just seqenced a couple of peptides from a tryptic digest of a PVDF blot. The
>sequences the "customer" wanted were at about the 10-15 pmole level. In one of
>the peptides a second sequence starting at about the 3 pmole level was easily
>read out to twelve amino acids. This second sequence turned out to be a
>tryptic peptide from keratin. So my question is where did it come from, skin?
>As I told the customer "I wore gloves".
>I recall seeing this a couple of years ago and chalked it up to someone's poor
>technique and forgot about it.
>I wonder how often this happens?
>Has any one sniped off a chunk of skin and processed it for internal
>sequencing?
>
>Steve