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Anion exchange purification


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#1 tasos

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Posted 14 November 2011 - 02:53 PM

Dear all,
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

#2 protolder

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Posted 20 November 2011 - 11:35 PM

Hola aggregates elute from Ni-agarose at high imidazol concentration above 0.3M. In may lab we dialyze Imidazol to avoid interferences before a ion exchanger but isn´t necessary concentrate because you could induce precipitation. Other possibility coud be made first ion exchanger pool active fractions and made the Ni.-colum withouth any treatment because it supports high salt concentration. Check on line quiagen 6His manual (Quiexpessionist) where you found incompatibilities of Ni-supports and where they recomend phosphate to Tris or other amines tertiaries and secondaries for buffer. Buena suerte

#3 u03cka

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Posted 07 December 2011 - 06:16 AM

I agree that you don't need to conc your protein before loading on to the column, especially resource as you can do a fast flow rate. You need to optimise your binding concentration as far as binding goes. You can vary the salt and pH until you find one that works.
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

#4 tasos

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Posted 08 December 2011 - 01:54 PM

My sample has the same ph as the start and elution buffer because I dilute it so I can get rid of the NaCl and the I'm using a superloop to load my sample which is 50ml.Additionaly i am always charging my column before equilibrating.Thank you for your replies and advices , I'll certainly try to experiment a little bit with different ph conditions.




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