Hello Christine,

We have done it before and it worked alright. We diluted the medium 1:3 with binding buffer. However, dealing with hundreds of milliliters of diluted LB medium was an issue. We therefore precipitated out total protein using ammonium sulfate, dialyzed against binding buffer and incubated with cobalt resin.

Hi there,

The crucial thing is to work at pH above histidine pK (usually 7.5 is OK) otherwise no interaction between His tagged protein and NiNTA  occurs. Diafiltration may be used to diminish the volume prior to purification.

 If precipitation of the protein is an issue, you could consider using Amicon centriplus centrifugation filter devices (or something similar) to first concentrate the extracellular proteins in the medium. There are various molecular weight cutoffs that you could consider. Once you have the protein concentrated and the volume reduced, then bring up the sample to the desired volume with your column binding buffer. Then purify the tagged protein per your protocol.