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Anion Exchange Buffers

Anion Exchange

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#1 Smog187

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Posted 26 February 2015 - 08:33 PM

Hello all, 

 

I am about to use our Anion Exchange Column to further purify my protein. 
We purified our protein using the His column, so it has 150mM NaCl, 150mM Immidizole and 20mM Tris. 

There are a few contaminating bands along with our eluted protein, so we are going to run it over our MonoQ column. 

 

My question is, I know that our protein will bind to the anion exchange column and that using a salt gradient will wash off proteins. 

 

But when we load our sample there is already salt present in our buffer. Will this salt affect our protein binding? Another lab suggested diluting down our sample to below 100mM salt concentration by adding starting buffer. 

 

Also the protocol says to wash 5-10CV with starting buffer (20mM Tris pH 8.0). However there is no salt in this buffer, and since salt is important to help solubilize proteins wouldn't this cause stuff to precipitate out of solution and clog our column? 

 

Thanks. 



#2 mdfenko

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Posted 27 February 2015 - 07:16 AM

the salt may interfere with your protein binding. you should load in buffer as close to "starting" buffer as possible.

 

i used to start with a relatively high salt concentration because i knew that my protein of interest would bind and a lot of contaminants wouldn't at that concentration.

 

as for destabilizing the protein at low salt, it depends on the protein. some require high salt to be soluble (e.g. myosin). some will aggregate at high salt (e.g. actin). most will precipitate at very high salt.

 

if you don't already have it, you can download a very useful handbook on ion exchange chromatography (amongst other useful handbooks from this ge lifesciences webpage.


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#3 labtastic

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Posted 27 February 2015 - 08:12 AM

All of your worries are distinct possibilities, but they may not be. Every construct of every protein is a different beast, so there is no good way to know up front what problems you will run into.

 

Some things to can do with your nickle eluted protein to test how sensitive it is to low/high salt:

 

Dilute concentrated protein in a no salt-buffer...let it sit for a little while and see if any precipitates

Dialyze into a no/low-salt buffer overnight...see if it crashes out

Desalt the protein using small disposable columns (eg PD-10, NAP-5 columns) into a no salt buffer...see if you get all your protein back out and if so, let it sit and see if any crashes out over time

 

If it looks like the protein is stable enough in no salt, then it should be ok to dilute into no/low salt buffer to bind to and wash on an ion exchange column

 

If your protein is not stable in low salt, titrate up the salt and see what the minimum is. I've worked with some proteins that absolutely require >500mM salt to stay soluble, and one I worked didn't require any salt at all and in fact was stable and fully folded in 50% ethanol after eluting from a phenyl sepharose column. You just have to test and see.







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