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Problems with Preparative Isoelectrofocusing (minirotofor)


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#1 villaca

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Posted 03 August 2010 - 05:44 AM

Hello everyone,
I have started to use the preparative isoelectrofocusing in non-denaturing conditions to purify IFNalpha overexpressed in E.coli as previous step at Affinity Cromatography.
I have put 20 mg of total protein, more or less, and I try with and without prefocusing. In the sample buffer (10 mM PB, 10 mM NaCl after dialysis) I added the ByoLytes 3-10 (4%), glicerol (up to 20%) and urea (up to 8M).
The problems that I am having is that I get up to 12 fractions between pH 6 and 8.9 (I think that this is not the right gradient), so my protein is not fine focusing and I have a little fraction of the protein that precipitated, and I am not sure that I can add detergent without loosing protein activity.
If someone could give some advice, I would be very grateful, I am a little lost.
Regards
Villaca

#2 mdfenko

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Posted 03 August 2010 - 06:58 AM

urea is denaturing.
talent does what it can
genius does what it must
i do what i get paid to do




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