Steven,
We currently use protein extractions as described by Molloy, Herbert, Walsh,
et. al (1998) Electrophoresis, 19, 837-844. The Sequential Extraction
Buffer #3 is very good for solubilizing membrane/hydrophobic proteins. Our
results show very nice, consistent gels using this extraction. I have not
stained the IPG strip post-second dimension to see how much protein is left
in the strip, so I can not comment on that possibility.
Jim Lawrence
Pioneer Hi-Bred Intl.
7300 NW 62nd Ave
Johnston, IA 50131
515-270-5909
Keep your head up and your stick down.
> -----Original Message-----
> From: Steven White [mailto:swhite@bmb-fs1.biochem.okstate.edu]
> Sent: Wednesday, November 15, 2000 2:25 PM
> To: Recipients of ABRF List
> Subject: P2D
>
>
> Hello all -
>
> A collaborator comments on the challenge of membrane proteins
> on 2D gels:
>
> Right now, I
> found the right conditions (detergents and others) for
> membrane proteins
> solubilization, I got really nice IEF gels. The problem is
> that most of the
> proteins (about 90%) do not transfert from the first
> dimension (the IPG
> strip from Biorad) to the second dimension (SDS-PAGE). It
> seems like their
> are interactions (maybe hydrophobic) between the IPG strip
> matrix and the
> membrane proteins. I used TBP as a reducing agent and I am
> wondering if
> this is not the problem. I am right now thrying several controls with
> either DTT or TBP as reducing agents. I hope I will solve this problem
> soon. This is extremely time consuming. Any idea?
>
> Can anyone comment on the above situation?
>
> Thanks,
>
> Dr. Steven P. White
> Oklahoma State University
> Department of Biochemistry & Molecular Biology
> 246 NRC
> Stillwater, OK 74078
>
> Phone & Voice Mail (405) 744-6191
> Fax (405) 744-7799
> e-mail swhite@biochem.okstate.edu
>
>
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