[Babcock_Hall]

I am familiar with some kinds of protein chromatography, but I now have to run a nickel column for the first time.  The protein of interest bears a histidine tag, and uur protein is believed to be a dimer or possibly a tetramer of identical subunits.  As is typical the nickel column is the first step after sonication of E. coli cells.  How do I choose the best volume of gel to use?  If I use too little, there will be loss of the protein in the load and wash.  If I use too much, the protein is more dilute, and in some sense I am wasting gel.

At first glance I can see that one issue is the need to estimate what fraction of soluble cell protein is the protein of interest.  I might be tempted to estimate this as being no more than 20% of the soluble protein.  A second issue is capacity of the gel, and based on my general knowledge of protein IEX chromatography it occurs to me that there might be some variation from one protein to another, based upon accessibility of the his tag.

[Babcock_Hall]

According to the manufacturer, the resin binds 5-10 mg of protein per mL of gel.