Hi Chris,

To get better and reliable results cleave the tags on both and use microscale thermophoresis(MST) or Isothermal calorimetry(ITC) or Biacore. Depending on the size one can use HSQC NMR too. But NMR experiments need 15N labeling. These methods give you quantitative results as well.

If your proteins degrade or are somehow unstable after cleavage then resort to on-column methods. As you have found, the on-column methods (immobilized methods in general) require a great deal of controls to be run, not only to convince yourself, but also skeptical reviewers later. If you can purify your proteins after cleaving the tags, you will get very clear results.

Hope this helps   

Thank you Dr. Ithychanda,

I figured cleaving the tags would probably be my next step to double check results.

I am not familiar with MST. Did you have any more information?

http://www.nanotemper-technologies.com/technologies/mst-technology/

Hi Chris,

Check this company site. They make the instrument. The reason you may want more than one technique is that sometimes you will run into technical issues that are tricky to resolve. Eg;No thermal signature in ITC or size limitaions of NMR. I have done ITC and NMR extensively. MST is sort of new and I saw a demo and was impressed. For some proteins-ligand combination where ITC failed, MST seemed work well. MST has its limitations too.

Sujay

Chris, you are seeing standard and common background interactions.  The purification columns are typically made out of sticky agarose/sepharose materials which, truth be told, bind to everything to varying degrees.  You want to make sure you have conditions where you can separate out signal from noise (there will always be noise) and there are many ways to do this: Try preblocking your column with BSA, or use less protein, or use less beads (column) or increase the amount of NaCl in your buffers.  In my experience a combination of all of these works best- Just make sure you don't put too much NaCl that you inhibit the interaction between your two proteins.   

Thank you Dr. Jordan,

My NaCl concentration is already decently high (from what I've been told) at 300mM. Preblocking is a great idea though. I will definitely be trying this. Thank you for the insight.