IEF migration to middle of gel, no separation - (Mar/01/2012 )

Hi

Im using the Phastsystem from GE Healthcare and im using pre-cast gels with a gradient 4-15

my problem is when i use the program designed for my kind of IEF (1D)
all the proteins migrate to the middle of the gel, regardless their pI.

I've started using different proteins as a reference after repeatedly failing with marker (out of date)
but Cyochrome C (pI10.7) goes just as far as trypsin inhibitor (pI4.5)

Im using these settings:
sep 1.1 2000V - 2.5mA - 3.5W - 15C 75Vh
sep 1.2 200V - 2.5 mA - 3.5W - 15C 15Vh
sep 1.3 2000V - 2.5mA - 3.5W - 15C 410Vh


If you have some tips please let me know

you are not using a gel suitable for ief. ief gels are not gradients, they are homogenous acrylamide concentration.

the phastsystem uses gels specific for ief.

mdfenko is right, it doesn't sound like you're using the right gel.

IEF gels have a pH gradient (typically narrow range like 4-7 or a broad range like 3-10) but are otherwise a low (around 5% non-gradient) acrylamide gel. They're usually bought pre-soaked with ampholytes, which define the pH range, or you can soak them yourself.