2D PAGE - reproducibility
dbell at qnis.net
Fri Nov 17 13:08:30 EST 2000
We just had a technician spend 1 - 2 years optimizing our 2DE protocol.
She found that different electrophoresis protocols worked for different
size IPG strips (i.e. 7 cm vs the 18 cm).
Pharmacia has a good discussion group that was very helpful, if you go to
their web page you should be able to get onto that group.
Use a set-up with cups to load the sample proteins onto the IPG strips -
the setup where you soak the whole strip in a reservior with your prep did
not work consistently. We bought a multiphor set-up from Pharmacia.
18cm strips 300V 5mA 20W 2hrs
1000 5mA 30W 2hrs
2500V 5mA 40W 2hrs
6000V 5mA 50W 130,000volt hours
7cm strips 200V 2mA 5W 5 - 10 min
gradient 200 - 3500V " " 1.5hrs
3500V " " 20hrs
One of my supervisors has forwarded your name and e-mail address to an
associate in New Zealand who spent some time training with us. Hopefully
she will get back to you and be a very good resource.
hope this helps
Molecular Markers Lab Technician
USDA Agricultural Research Service
2021 S. Peach Ave.
Fresno, CA 93727
e-mail: dbell at qnis.net
phone: 559 453-3170
fax: 559 453-3088
> From: Gerhard Schwall <gschwall at iuct.fhg.de>
> To: methods at hgmp.mrc.ac.uk; proteins at hgmp.mrc.ac.uk
> Subject: 2D PAGE - reproducibility
> Date: Wednesday, November 15, 2000 6:42 AM
> Currently I am doing 2D gels using purchases IPG strips in a Protean IEF
> camber from Biorad.
> I have tremendos variations in my results, even when I run several
> strips with exactly the same sample in a single run.
> A few gels look absolutely ok and others completely unfocused.
> I tried already different things like passive/aktiv rehydration,
> with/without electrode wicks etc. The result is always the same:
> Some gels are totally allright, others are completely unfocused. All
> strips a treated exactly the same!
> I am wondering if anybody had the same problems or might have a
> suggestion what might cause the trouble?
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