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IEF migration to middle of gel, no separation

IEF protein

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2 replies to this topic

#1 Janthara



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Posted 01 March 2012 - 07:29 AM


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I've started using different proteins as a reference after repeatedly failing with marker (out of date)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

#2 mdfenko


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Posted 01 March 2012 - 12:26 PM

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.
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#3 ks.ca



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Posted 14 May 2012 - 09:55 AM

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.

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