Problems with Preparative Isoelectrofocusing (minirotofor) - (Aug/03/2010 )
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.
urea is denaturing.