problem with ion exchange puification - protein not binding (Aug/27/2010 )
hi,
I am purifying HIV Gag (55 KDa)HIS tagged protein , bacterial expression.I get the protein at all concentraions of imidazole 100 mM, 200, 300 and 500 Mm(in phosphate buffer pH-6.4 ) .MY homogenization and wash buffer has 20 mM imidazole.i want to very pure protein as it is for immunological work.So I further try purifying it by Ion exchange using Sp sepharose beads.The pI of my protein is 9.but my protein is not binding to the beads.I directly apply the NI-NTA elutes on beads equlibrated at pH 6.4.I have also tried equilibration of beads at defferent pH from 5.8 to 7.8. But there is very less binding.
please give me some solution.
Hola, yes this is due to the imidazole presence in your sample wich interacts with ion exchanger avoiding the bounding of your protein, DYalise or better dilute ten fold your sample and repeat ion exchanger. I had in my bench a paper, but I dont find it to see the reference.Good Luck
protolder on Fri Aug 27 09:50:53 2010 said:
Hola, yes this is due to the imidazole presence in your sample wich interacts with ion exchanger avoiding the bounding of your protein, DYalise or better dilute ten fold your sample and repeat ion exchanger. I had in my bench a paper, but I dont find it to see the reference.Good Luck
thanks a lot. I have diluted it four times. i ll try after dialysing it.
shilpeejnc on Fri Aug 27 15:26:39 2010 said:
protolder on Fri Aug 27 09:50:53 2010 said:
Hola, yes this is due to the imidazole presence in your sample wich interacts with ion exchanger avoiding the bounding of your protein, DYalise or better dilute ten fold your sample and repeat ion exchanger. I had in my bench a paper, but I dont find it to see the reference.Good Luck
thanks a lot. I have diluted it four times. i ll try after dialysing it.
If you could find the reference then kindly send me, so that I can show it to my PI.