You can find answers on internet sites of companies involved in the matter, such as Bio-Rad...

Look here:

https://www.bio-rad.com/fr-fr/applications-technologies/anion-exchange-chromatography?ID=MWHAZ4C4S#3

To do a quick experiment, condition your resin with 1 M NaOH for 5 minutes to load up the anion exchange sites with OH-. Equilibrate the resin in water for 5 minutes to remove any unbound OH- . Since your protein has a pI of 9.73, you will have to load in a buffer that is at least 10.7 so that it is in its negative form, but use a low concentration buffer so that the buffer anions do not take all of the resin binding sites. If you want to do a wash, you have to keep the pH above 10.7, but again use a low concentration so that the protein anions are not exchanged. To elute, you can either use a lower pH buffer (pH~6) so that the protein changes to its cation form and no longer binds to the column, or use a high salt solution (0.8M) to displace the protein with the salt cations. It just depends on what you want to do next with your protein

Pharmacia/LKB had a very useful free manual on IEC ("Ion Exchange Chromatography Principles and Practice"). They were later bought by Amersham which was bought by GE. Perhaps your rep can get you a copy.

Waters Corporation also has an excellent guide to solid phase extraction called [Beginners guide to] SPE. You can contact them for a copy.

Lots of ways to go about this and unfortunately protocols can be protein specific. Are you expressing from E.coli or Mammalian or other ? Is it secreted of cytosolic ?

Anion exchangers have a positive charge and depending on resin used can hold their charge over a broad pH range.They therefore bind proteins with a net negative charge. Your protein has a pI point of 9.7 which basically means below this pH it will have a net positive charge and above this will have a net negative charge. This may mean that you have to have a pH above 9.7 in order for your protein to bind to an anion exchanger (please note it is not always that simple). I would carry out small binding study to assess binding of your protein and contaminants.

1. pH range 6-10 (I have no idea about the stability of your protein at higher pH's but if stable you can increase pH). Go up in increments of 1 pH unit to start with i.e. 6, 7, 8, 9, 10 etc. Then if successful 0.5 pH unit. Choose buffers that can cover this range: see many protocols on this (GEHealthcare for example). Check stability of protein at each pH: incubate for 10 minutes in pH buffer then spin 20k g. Check supernatant/pellet for protein loss.

2. Equilibrate~50 uL of resin in the appropriate buffer above in eppendorff. Use approximately 2 x 1 ml buffer. Spin and remove buffer carefully with pippette.

3. Add appropriate volume/amount of target protein. What you want is to be able to detect your protein/contaminants on an SDS-PAGE gel to visualise (you may need to test this initially). What you are looking for is either loss of your protein from solution due to binding resin OR loss of contaminating protein. Mix 5 minutes on roller rig or manually every 1-2 minutes (gentle mixing).

4. Spin and remove supernatant for analysis (Protein con/SDS-PAGE).

5. Wash resin with pH buffer (1-2 mL as previously described).

6. Add equal volume of 1M NaCL in pH buffer (Just add 1/5th volume of 5 M NaCl to appropriate buffer) and mix for 5 minutes as before. Spin and analyse protein.

Note: it is highly unlikely that you will get a pure clean protein using a 1 stage purification like this but you never know. If you are purifying from E.coli then the high pI point is a big advantage since most E. coli proteins have pI points around 5 so a cation exchanger would be good.

With an anion exchanger what you might look at is trying to bind contaminating proteins and look for your protein in the unbound fraction. At pH 8 most proteins (Except yours) will have a net negative charge and bind the anion exchanger, your protein will pass through.

There are other techniques that could also be useful at lab scale if equipment/materials are scarce

1. Ammonium Sulphate precipitation

2. pH precipitation

It is also important to understand if the resin you have is a strong anion exchanger (charge on resin is independent of pH) or a weak anion exchanger (charge is pH dependent), as that defines both loading conditions and elution conditions.

Jerome P Ferrance

Can I condition a weak anion exchanger (working pH is 2 - 7) using 1 NaOH ?

Thanks

Chandan, No, this is not the way to do things. For a normal weak anion exchange column, you would want to keep the pH less than 7 or else the column will lose all of the associated charge and not capture anything.

For conditioning the column, you can use either HCl or NaCl at low pH. This will fill the anion exchange sites on the column with Cl- ions. Rinse the column with neutral water, then load your solution in slightly acidic (pH ~5) solution. Your anions should displace the bound Cl- anions. Wash the column again with water to remove any unbound material. Elute by raising the pH above about 8, which will remove the + charges on the column and release the captured anions.

You can also elute using a high NaCl concentration, but the advantage of the weak exchangers is that you can change the charge on the column with pH and do not have to use high salt to elute.

General protocol for using an anion exchange resin, such as DEAE-Sepharose, Q-Sepharose, Capto Q, or other strong/weak anion exchangers, applicable for biomolecule purification.