His Tag purification - specificity problem (May/27/2005 )
Dear all
hi i am working on a protein
i have cloned it in pQE 32 which give a protein with His tag at N-terminus and when i run affinity i got single and sharp peak but when i run it on SDS PAGE it gives multiple band that include the band of my intrest also
what should be done to increase the specificity of affinity purification.
your early response will be very helpful for me
Niks
His taq- Ni sepharose is not as effeceint as described by the catalogues. The problem is that there are metal binding proteins which too append to the column and elute lateron. Try gel filtration after the first column. Thats what we do for one of the his tag clone in our lab.
What's your purification condition? If your protein is sticky and copurified with other contaminants, it won't show up as multiple peaks in chromatography but it will when denatured and reduced. So perhaps you want to add detergent (triton, urea) and high-salt (300 mM to 1 M NaCl).
Good luck,
Lightbulb
Good luck,
Lightbulb
hi
i am already adding 1% triton X and salt conc. i am using is 0.5 M Nacl
eluting with gradient from 20mM - 500Mm .
Are you suing Ni-NTA or Ni-TED? Ni-NTA results in non specific binding which is eliminated substantially using Ni-TED resins. Good Luck! 
hi bassam
can you please tell me wht is Ni-TED resin from which company .
i am sorry if it sound like a silly question.
Thanx
The companies that I know hey have the resin available are Active Motif, Machery-Nagel, and if you google search it you will find more. Good Luck!
Hi, does Ni-NTA specificity depend on whether the protein is being purified in native vs. denaturing conditions? I purified in denaturing conditions and I only got one contaminating band.
However, I purified under native conditions and I have many more contaminating bands. Based on this, do you think I should make the switch to Ni-TED?
hi i am working on a protein
i have cloned it in pQE 32 which give a protein with His tag at N-terminus and when i run affinity i got single and sharp peak but when i run it on SDS PAGE it gives multiple band that include the band of my intrest also
what should be done to increase the specificity of affinity purification.
your early response will be very helpful for me
Niks
Hi Niks,
Most likely you need to optimize a bit on the elution and also the concentration of imidazole in your sample & binding buffer. Can I ask you a few questions:
1. Are you running this in a fplc system or manually?
2. Is it a step gradient or a linear gradient?
3. Is there any imidazole in your sample buffer and binding buffer?
Henry