I don't have personal experience, but I can tell you that the presence of a high concentration of histidine is the only thing in the medium recipe that could cause trouble. However, the 100X histidine solution is only about 26 mM, so at 1X (0.26 mM), it would not be an issue.

Adam B Shapiro , good point, however I should have indicated that I am using the strain X-33 so I won’t need to supplement with histidine. I am primarily worried that media components could outcompete the protein for binding as you suggest. Thanks for your thoughts!

High salt concentration to minimize electrostatic interactions is a must in imac; as well as a pH value above 7.

Thomas J Esparza Just curious if you tried this without the TFF exchange. In your recent publications you used TFF, I just wanted to follow-up and see if there was a reason behind it. Thanks in advance!

Ryan Garrigues thanks for the follow up. This now seems like ages ago, with the craziness of covid, but still good to address. For our paper we outsourced the fermentation to achieve a scale sufficient for the animal study. Our CRO at the University of GA did use TFF to concentrate the supe prior to Ni-NTA purification. We also purchased a small scale TFF system (MiniMate from Pall) not long after posting this question and it works great. However, I have found that by simply added 10xPBS to the media and supplementing with NaCl to a final 0.5M concentration that I am able to directly run the media over a HiTrap column using a sample pump to purify same day. The limitation is the flow rate of the column, so I tend to grow no more than 1-liter at a time. I have used this system to successfully purify nanobody constructs and scFvs with excellent initial purity and then polish them by SEC.

Best,

Thomas