Greg,
I have not used these dyes myself, but I have looked at them carefully,
because the basic idea is a good one (at least I think so). However, there
are some problems that Amersham Pharmacia Biotech had still not licked when
I last talked with them (6 months ago), so I am not very enthusiastic right
now.
The dyes are used to covalently modify proteins prior to 2D, but at a very
low level, in order to make the quantitation work. This would also avoid
problems with later chemistry, but it has a down side. Under-labeling
results in a difference in electrophoretic migration in the second
dimension. Unlabeled material does not co migrate with the protein that has
been labeled. This effect can be pretty large for a small protein, and it
is not unlikely that one could completely miss the majority of the material
when cutting a spot, or get some other unlabeled protein(s) migrating under
your visible spot. Of course, co-migrating proteins are not a new problem
in 2D gels, but missing material might very well create significant
sensitivity issues, and this entire situation complicates an already
challenging process.
So far as I know, APB is working on new chemistry that will permit
quantitative derivatization of proteins, obviating the need for under
labeling, and also, of course, eliminating the differential migration
problem. Again, my information is about six months old, so I don't know
where this stands.
Hope this helps,
Doug Sheeley
Views expressed are my own, and do not reflect a position of NCRR or NIH.
Douglas M. Sheeley, Sc.D.
Division of Biomedical Technology
National Center for Research Resources
National Institutes of Health
6705 Rockledge Drive
Bethesda, MD 20892-7965
Voice: (301) 594-9762
Fax: (301) 480-3659
Email: sheeleyd@ncrr.nih.gov
-----Original Message-----
From: Greg Grant [mailto:ggrant@pcg.wustl.edu]
Sent: Monday, April 16, 2001 12:10 PM
To: Recipients of ABRF List
Subject:
Dear Colleagues,
Can anyone provide any information on the Cy 3 and Cy 5 dyes being
marketed by Amersham as to their properties and compatability with mass
spec analysis. Like, how do they interact with proteins? How sensitive are
they? Do they need to be removed prior to analysis? and anything else.
Thanks,
Gregory A. Grant
ggrant@pcg.wustl.edu
314-362-3367
FAX 314-362-4698
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