Hey Robert, what kind of beads do you use in the first step? I had a similar problem - I got advice to do washing steps with ATP (didn't help), but switching from Ni-NTA/Ni-IDA beads to Talon beads did, because these contain cobalt instead of nickel.

Hi Robert,

SlyD is a common E.Coli contaminant in Ni-IMAC, but protein expression appears to be the problem here.

You should consider using a different vector that incorporates a His-fusion tag (SUMO, GST, MPB) to your protein. These tags can improve expression and solubility. You can cleave the tag off after the first Ni-IMAC, dialyze your protein back to binding buffer and perform a second Ni-IMAC to remove the His-fusion tag and SlyD. Your protein should elute out in the flowthrough/wash.

Hi Robert,

You might use another approach, for example, you can use an anion exchange chromatography after the IMAC step. The isoelectric point may be substantially different between the overexpressed protein and SlyD.

Best regards

Hi Robert,

Here's a recent article with an example of SlyD contaminant removal by affinity chromatography:

http://www.sciencedirect.com/science/article/pii/S1046592817301651

Hope this helps,

Eric