Hi This is a multidimensional problem, just like any other tricky protein purification.
1) IMAC columns are also ion exchangers (of both polarities!) so make sure you have adequate salt. 2) IMAC resins can be polysaccharide-based (agarose) and affinity to sugars can be an issue. Consider adding a 'mock substrate' (basically a high level of some inexpensive sugar). 3) Even if you cannot use IMAC there are other options, people purify native proteins in a 'generic' manner all the time :) 4) definitely a case for trying other resins. Especially consider resins with pentavalent chelation - they have rather different properties from resins based on tri (IDA) or tetra (NTA) chelators. 5) as others have mentioned, you may have a problem of the protein as such - can you assess aggregation state before purification (e.g. SEC of clarified lysate followed by activity assay). Artem - Cosmic Cats approve of this message On Fri, Sep 15, 2017 at 7:53 AM, Narayanan Ramasubbu < [email protected]> wrote: > Hi. We are working on a periplasmic protein that breaks naked glycans in > peptidoglycans. There is truncated structure available but our target is > the full length protein. The difficulty us that it strongly binds to the > resin with or without his.tag. Changing the resin to acrylamide did not > help. > Has anyone come across similar problem and how was it resolved. > The pdb structure is the catalytic domain and mussing a region that, in my > opinion, binds to the resin. > Thank you in advance > Sent from my iPhone
