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Derek:
Dodecyl maltoside instead of triton X-100 (NP-40) or CHAPS has worked well
for delipidation and resolving membrane proteins in liver homogenates
[Dodecyl maltoside detergent improves resolution of hepatic membrane
proteins in 2D gels. Electrophoresis 12, 687-688, 1991]. You might try it.
Good luck,
Frank
*************************************
Frank A. Witzmann, Ph.D.
Professor of Biology
Molecular Anatomy Laboratory
IUPU Columbus
4601 Central Avenue
Columbus IN 47203
Phone 812-348-7215
Fax 812-348-7279
http://iupucbio1.iupui.edu/frankw/witzmann.htm
*************************************
-----Original Message-----
From: Association of Biomolecular Resource Facilities
[mailto:abrf-request@aecom.yu.edu]On Behalf Of Derek.Ellison@Medeva.com
Sent: Thursday, December 02, 1999 10:05 AM
To: Recipients of ABRF List
Subject: 2D-PAGE problems
Dear ABRF,
I am trying to develop a 2D page assay to separate, identify and possibly
quantitate the components of a macromolecular protein complex. The
component proteins are extremely hydrophobic with several membrane spanning
domains and several are glycosylated. The complex also has associated
lipid.
So far we aren't having much luck, lots of streaking (despite delipidation),
extremely poor reproducibility basically due to the pretty poor solubility
of the proteins in the common non-ionic 2D detergents we've tried.
At a meeting i was at recently, one of the speakers mentioned (in passing) a
technique that i think he called zone (zonal?) electrophresis and put up a
2D like gel. The inference was that if you couldn't get the hydrophobic
proteins you were interested in to run on standard 2D you could use this
weaker resolution technique which had the advantage of being able to use SDS
in both dimensions. The only zone electrophoresis i've ever heard of is CZE,
and this certainly wasn't it. I may have misheard, but in that case so did
the guy sitting next to me as we both agreed on what was said. Anyone out
there know what I am babbling about?
Alternatively - anyone got any suggestions for some good, novel detergents
to try and where to get them?
thanks for the advice,
Derek Ellison
Medeva Development
Speke
Liverpool
L24 9GR
Phone: 44 151 705 5442
Fax 44 151 705 5189
This email contains confidential and proprietary information some or all of
which may be legally privileged. It is for the intended recipient only. If
you have received this email in error, please notify the author immediately
by telephone or by replying to this email. If you are not the intended
recipient you must not use, disclose, distribute, copy, print, or rely on
this email.
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Derek:
Dodecyl maltoside instead =
of triton=20
X-100 (NP-40) or CHAPS has worked well for delipidation and resolving =
membrane=20
proteins in liver homogenates [Dodecyl maltoside =
detergent improves=20
resolution of hepatic membrane proteins in 2D gels. =
Electrophoresis 12,=20
687-688, 1991]. You might try it. Good=20
luck,
Frank
************************************=
*
Frank A. Witzmann, Ph.D.
Professor of =
Biology
Molecular Anatomy=20
Laboratory
IUPU Columbus
4601 Central Avenue
Columbus IN=20
47203
Phone =
812-348-7215
Fax =20
812-348-7279
***=
**********************************
-----Original Message-----
From: Association of =
Biomolecular=20
Resource Facilities
[
Sent: Thursday, December 02, 1999 =
10:05=20
AM
To: Recipients of ABRF List
Subject: 2D-PAGE =
problems
Dear=20
ABRF,
I am trying to develop a 2D page assay to separate, identify =
and=20
possibly
quantitate the components of a macromolecular protein =
complex.=20
The
component proteins are extremely hydrophobic with several =
membrane=20
spanning
domains and several are glycosylated. The =
complex also=20
has associated
lipid.
So far we aren't having much luck, lots =
of=20
streaking (despite delipidation),
extremely poor reproducibility =
basically=20
due to the pretty poor solubility
of the proteins in the common =
non-ionic 2D=20
detergents we've tried.
At a meeting i was at recently, one of =
the=20
speakers mentioned (in passing) a
technique that i think he called =
zone=20
(zonal?) electrophresis and put up a
2D like gel. The inference was =
that if=20
you couldn't get the hydrophobic
proteins you were interested in to =
run on=20
standard 2D you could use this
weaker resolution technique which had =
the=20
advantage of being able to use SDS
in both dimensions. The only zone=20
electrophoresis i've ever heard of is CZE,
and this certainly wasn't =
it. I=20
may have misheard, but in that case so did
the guy sitting next to me =
as we=20
both agreed on what was said. Anyone out
there know what I am =
babbling=20
about?
Alternatively - anyone got any suggestions for some good, =
novel=20
detergents
to try and where to get them?
thanks for the=20
advice,
Derek Ellison
Medeva =
Development
Speke
Liverpool
L24=20
9GR
Phone: 44 151 705 5442
Fax =
44 151=20
705 5189
This email contains confidential and =
proprietary=20
information some or all of
which may be legally privileged. It =
is for=20
the intended recipient only. If
you have received this email in =
error,=20
please notify the author immediately
by telephone or by replying to =
this=20
email. If you are not the intended
recipient you must not use,=20
disclose, distribute, copy, print, or rely on
this=20
email.