Anion exchange purification
Posted 14 November 2011 - 02:53 PM
I am trying to purify a protein which has a pI of 5.17. It has a His-Tag so after a Ni-column I am trying using a ResourseQ anion exchange column. My protein after the Ni column is in a salty solution 300mM NaCl+50mM Tris+Imidazol+glycerol so I first concentrate it down to 200microliters and then dilute it to my start buffer(50mM Tris pH 7.5) up to 50ml to lower the salt concentration. The elution buffer I use is a 1M NaCl + 50mM Tris pH 7.5 so in these conditions my protein should be negatively charged and be able to bind to my column but I dont get anything, the protein doesnt bind and I find it always in the waste. Could it be that is aggegated even before applying it to the column. I have to mention that if after the Ni column I procced with Gel filtration column then I get some protein but its not pure enough for me. And one last thing for a similar protein(same family-size-pI) the same anion exchange column works fine and thats the most frustrating thing! Any ideas?
Thanks in advance
Posted 20 November 2011 - 11:35 PM
Posted 07 December 2011 - 06:16 AM
Remember, you need to charge the column first by running a couple of CV of you elution buffer through, before equilibrating in binding buffer. Also, your sample pH must be the same as you binding pH for optimal binding.
PS - cut your tag off and rebind with nickel = purer protein
Posted 08 December 2011 - 01:54 PM