2D PAGE - reproducibility

Deanne Bell dbell at qnis.net
Fri Nov 17 13:08:30 EST 2000


Hi
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).
In general:
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
Deanne Bell
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?
> Regards
> Gerhard
> 
> 
> 
> 
> 


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