2 replies to this topic

[Janthara]

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I've started using different proteins as a reference after repeatedly failing with marker (out of date)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

[mdfenko]

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.

[ks.ca]

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.