Iso-Electric Focusing Questions

Joan Shields joan at med.unc.edu
Thu Apr 22 13:21:58 EST 1993


I'm having problems with my IEF!!

Let's see: I've been pouring my own gels and they seem to be pouring fine
but when I run them I only get staining (and not very good) on one end
(right-hand side) of the gel.  I'm using a flatbed set-up, power: 4 watts,
9 (starts at 9 and goes down to about 3.5) miliamps, and about 1000 volts 
(starts at about 400 and goes to 1000).  I'm using a mix of ampholytes:
5.0-7.0 and 3.5-10.0 from Pharmacia.

1. Would a too low voltage cause the samples to run only on one side of
the gel?

2. Would a precast gel (5.0-6.5) be best?

3. How about pre-blended ampholytes? (Pharmacia makes a 5.0-6.5
pre-blended mix) 

4. How hard is it to successfully pour an IEF gel?  One gel I did worked
but I haven't had any luck since.

5. Anything I should look at that I haven't already looked at? (I've
checked the stain and it's ok, I've pour straight SDS/PAGE and gradient
gels that worked)

6. HELP!!!!!

7. e-mail would probably be best

Thanks,

Joan



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