I would attach to the Flag tag affinity resin, and then use that column to increase the salt concentration to save a step. If you know your complex stays together in low salt, wash the affinity matrix with low salt buffer. Then when no further protein comes off, increase the salt concentration to see if other subunits of the complex will come off. You can start, by first just making sure the affinity column will work to purify your intact complex. I don't understand why you need the anion exchange column, unless the protein is supposed to elite from the flag tag resin with salt. Does the flag tag not bind the resin under high salt?

Dear Tilak,

If you used ion exchange chromatography, you will release some of the subunit but it won’t necessary indicate that they are loosely bound. Protein interacting through electrostatic interaction will detach, but interaction promoted by hydrophobic interaction will bind even tighter. I would suggest the M2 affinity resin and elution with Flag peptide followed by Size Exclusion Chromatography. To find out which one are loosely bound youcould gradually increase the temperature of your purified complex and see when the subunit detach.

Best,

Laurent

Dear Marcia,

Thanks for your suggestion.

I tested till 300mM NAcl Conc and 3XFlag-tagged protein was bound to the affinity resin. 

I don't know up to what conc. I can go with NaCl without losing the integration of 3XFLAG tag with M2 Resin.

When I purify the protein complex of my interest with 3xFLAG-M2resin, I still get a lot of contaminants proteins. I wanted to use the Anion Exchange chromatography to make sure that the protein subunit which has the 3XFLAG tag, should be attached to the Anion exchange resin, while other contaminants and other subunits of the complex can be washed off by increasing NaCl Conc. 

Hi Tilak,

I don't know if its too late to reply.

Anyway you could use 5-10 mM ATP with 5-10mM MgCl2 in wash buffer. This will help to remove chaperones that might be bound to your target protein. These are typically the major contaminants during initial purification steps.