2-D Gel Problems with IEF

[kiki06]

Hey guys,
I am having problems with my IEF (isoelectric focusing). It seems the second dimension is fine, but the IEF is messing up somehow. I think it may be the voltages.

Does anyone have any recommendations for the voltages?

thanks
Kiki

[csteed]

Using Biorad Ready Gels I use the following settings:

100V 60 min
250V 60 min
500V 60 min

25mA Max
10W

I run the gels using NaOH in the inner chamber and Phosphoric acid in the outer chamber. Works well for me.

[protdoc]

what exactly is ur problem ...? make it little more elobarate...

[leaf]

Using the bio-rad IPG strip,for 17cm,passive absorption,i use the following step:250V 30 min
1000V 1h
10000V 5h
10000 60000Vh
500V anytime

[sivaramaiah]

Hi
Whts the programme you gave for IEF and wht type of fluctations you notice. write in detail
regards
SIvaramaiah

[alanamco]

As condições de corrida irão depender da faixa de pH e do tamanho da strip.
Em que faixa você esta utilizando???

[mlhdopamine]

Hi here is what im using, but mine isnt working either.
250V 15 minutes
4000V, rapid ramp, totaling 20000vh
500V hold step.