Old school 2D gel electrophoresis

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Mon Apr 28 14:17:02 EST 2008


Am 25.04.2008, 16:06 Uhr, schrieb Arne K Christensen <arnec from bio.umass.edu>:

> I am interested in doing some proteomic work using 2D gel  
> electrophoresis.  We have the hardware to run 2D gels, however our  
> equipment is many decades old and uses tube gels to resolve in the first  
> (IEF) dimension.  Most of the published in the last 10+ years describes  
> a first dimension separation by immobilized pH strips, which is  
> presumably better.

Yes, and for good reasons:

- the flat gels use immobilised pH-gradients, which do not show cathodic  
drift and work for protein with more alkaline pI. They are also more  
reproducible than the ampholine based ones.

- cooling is much more effective

- in traditional tube gels the protein is added at the acidic side. In my  
experience a lot of protein precipitates under these conditions and  
doesn't enter the gel at all. In flat gels you can add the sample at  
whatever pH you choose (or simply soak the sample into the gel during the  
rehydration phase)

- tube gels tend to break when you push them out of the tube (or do not  
come out in the first place). Flat gels have a plastic backing, which is  
almost indestructible.


> Does anyone in this group have experience using tube gels?  Does the  
> newer method truly warrant the costs associated with it (BioRad cost for  
> the power pack is ~$8,000)?

IMHO unequivocally yes, if you have to do it more than 2 or three times.  
But if you want to fiddle with the tubes (just to see how heroic those  
times were), at least polymerise a cotton thread into the gel for easier  
handling (Patton et al., Biotechniques 8 (1990) 518-27). Btw, I was a very  
satisfied customer of the Pharmacia IGPhor unit, no idea whether it's  
still on the market (Pharmacia was bought by GE).


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