Yuka,
If the problem is confined to the protein sequencing methods (i.e.,
ethyl acetate "chemistry", as opposed to acetonitrile/toluene "chemistry"),
then you may be using defective ethyl acetate. Water/peroxides in the ethyl
acetate will give a pronounced reduction in PTH-Lys yields and usually lowers
both IY and RY. For example, when a bad lot of ABI S2B (ethyl acetate) was
used to sequence 10 pmol of HSA with 3.1 chemistry, the IY was reduced by
half, the RY dropped about 1.5% and the Lys-4 recovery was reduced five fold
as compared to an HSA analysis using B&J BioSyn grade ethyl acetate. Please
note that in the past 2 years, bad batches of ethyl acetate from HP/Agilent,
ABI and B&J have been encountered here. So it is a good idea to be vigilant
regarding ethyl acetate quality, regardless of who sold it to you.
If your problem is not related to ethyl acetate, are you using any
reagents or solvents not manufactured by either Agilent or ABI? If yes, try
changing it/them to either an Agilent or ABI reagent, especially if you just
changed lot #s. By the way, there is nothing necessarily wrong with using
non-sequencing grade reagents/solvents. But if a problem arises when you
change from one lot to another, then it/they move to the top of the suspect
list.
Regarding your question about R1, R2 and R3, you can rule out R1 by
using ABI R1 diluted with heptane to 3% PITC (or just use undiluted ABI R1)
and rule out R3 by using ABI R3. To my knowledge, there is no commercial
substitute for HP/Agilent R2, but the historical problems with HP/Agilent R2
have usually been related to chromatogram artifacts, not bad chemistry.
And don't forget to watch an entire chemistry cycle (sample column and
flask) to verify that everything looks normal.
Hope that helps,
Steve
====================
Stephen Tindall, Ph.D.
Argo BioAnalytica, Inc.
====================
Subj: HP protein sequencer
Date: 10/29/2000 6:42:34 AM Eastern Standard Time
From: Yuka_Matsumoto@trc.toray.co.jp (Yuka Matsumoto)
Sender: abrf-request@aecom.yu.edu (Association of Biomolecular Resource
Facilities)
To: abrf@aecom.yu.edu (Recipients of ABRF List)
Dear All:
We have been using a HP G1000A Edman sequencer for many years. But now We
can't good an initial yield of PTH amino acid. I think that one of the reason
is reagent's quality,especialy the reagents related to Edman reaction
directly,R1,R2 and R3. So I will get other new reagents.
Are there any other reason for that problem?
Your responses would be greatly appreciated.
Yuka Matsumoto
Toray research center Corp.
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