I have been able to express a soluble 6xHis-tagged protein but purification is extremely inefficient. We are only able to increase our yield by overloading the resin (8-10 ml lysate on 50 ul resin), with the majority of tagged protein washed out in the flow through.

I suspect that our recombinant protein is forming soluble aggregates that impede purification; and only a small portion of non-aggregated protein is free to bind the Ni-NTA resin. Purification under denaturing conditions (8M urea) results in all protein binding the resin with nothing detected in the flow through.

Would running our soluble lysate on a native page followed by western blot reveal whether the protein is aggregated (i.e., detecting a band at a much larger size in addition to a band of the expected size)?

I have tried everything to improve purification (increasing salt concentration, adding imidazole to the lysis buffer, adding detergents to the lysis buffer, phosphate vs. tris buffer, reducing molarity of buffer, adding 0.25-5 mM DTT and 5-10 mM B-Me to the lysis buffer). We are not happy at the inefficiency of having to overload the resin but it is working. I would simply like to know if we could confirm whether our protein is forming aggregates by native PAGE.