Estimating the amount of nickel gel to use

[BabcockHall]

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.

[BabcockHall]

The manufacturer claims that 5-10 mg of protein binds per mL of resin.

[CharonY]

On 11/14/2020 at 1:52 PM, BabcockHall said:

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.

I do not think that this can be easily generalized, especially as there are different systems. I found that having a good packing (based on the geometry of the column) is more important than the resin to substrate ratio. However that is not really based on stringent testing. The bigger issue I see with a low abundance of the protein is that the column captures more unspecific metal-binding proteins.

[BabcockHall]

Would you please say a bit more about good packing and column geometry?  With respect to gradients and ion-exchange, I have seen a book the recommended short, broad columns over taller columns.  But I don't know whether or not this carries over to affinity columns.

[CharonY]

I mean that for a given column size you it works better to have sufficient resin to have an even packing, avoiding air gaps and so on. It depends a bit on whether you use syringes, spin- or gravity columns.

Thinking back, I think too much resin could encourage unspecific capture, as there is less competition with your tagged protein, so I may have to walk back that statement a bit. Unless we run into trouble (e.g. after quantifying which proteins we isolated using mass spec) we tend to use a standard amount which corresponds to usually more than we need. But we do not try to optimize it before each isolation.