ion-exchange chrom. under denature conditions - purification on IEX chrom from inclution body (Oct/19/2004 )

Hello everybody ,

I am wondering, if anybody knows techniques to purify protein on ion-exchange chromatography under denature or partially denature conditions?

I am purifying protein from inclusion bodies and after Ni-NTA in 6M guanidine hydrochloride it still not pure enough. Theoretically it should be possible to bind protein to ion-exchange media in 6-8M urea, isn’t it? I have already tried with SP-Sephadex G50 (Pharmacia) under 6M of urea. All protein goes in flow-throw fraction. Does anybody try something like that and which resin I should use?

Many thanks in advance,

Alexei

Hello everybody ,

I am wondering, if anybody knows techniques to purify protein on ion-exchange chromatography under denature or partially denature conditions? 

I am purifying protein from inclusion bodies and after Ni-NTA in 6M guanidine hydrochloride it still not pure enough.  Theoretically it should be possible to bind protein to ion-exchange media in 6-8M urea, isn’t it? I have already tried with SP-Sephadex G50 (Pharmacia) under 6M of urea. All protein goes in flow-throw fraction. Does anybody try something like that and which resin I should use?

Many thanks in advance, 

Alexei

Being a salt, guanidium chloride might be affecting the binding. Try with urea. Is it vital for you to run the sampe under denaturing condition.

good luck