We are following a paper in which they purified their histidine-tagged protein (same protein as ours) using a gradient of imidazole. However, they did not specify the volume of buffers. This affects the steepness of the gradient. What would be a logical choice? A Methods in Enzymology article from around 2000 by Barnhorst and Falke Reference suggested a 20-column volume gradient.

Edited June 13Jun 13 by BabcockHall

20x column is a rule of thumb for a mostly quantitative flush of the column. If running a gradient, it depends a fair bit on the purpose. E.g., whether you want to assess relative purity or optimize imidazole concentrations. The elution profile would roughly follow the same parameters as "normal" LC on parameters like peak width (related to flow rate and volume and gradient steepness), for example.

We would like to purify this protein away from others for kinetic and structural studies. We anticipate that there will be a buffer exchange step afterwards to remove the high concentration of imidazole.

Are you targeting a specific fraction using FPLC or similar? I generally only used gradients (way back) to optimize wash and elution conditions and then used those parameters for purification. If you have a FPLC setup rather than control for total volume you would go for an appropriate flow rate for your system in combination with the column to get decent-sized peaks of your desired fraction.

I probably do not understand your question. We were planning to use a classical gradient maker and gravity to run this column, as opposed to FPLC. However, we have access to an FPLC, and we have a few anion-exchange columns. I was thinking that this might be an appropriate second purification step, if one were needed. Cibacron Blue chromatography might also be a good second step. BTW, my reading of commercial literature on nickel-based IMAC indicated that some groups use a batch method. It is not clear to me what the advantages or disadvantages are.

Edited June 16Jun 16 by BabcockHall

Oh OK, I should probably explain my reasoning better. Since you mentioned a gradient elution approach, I assumed that you were trying to optimize the elution profile. There are various reasons that I am aware of to do so, including optimizing the imidazole concentration which then can be used for purification rather than running a gradient every time, it can be part of off-or online methods where you use a fraction collector to catch your fraction of interest and so on. As that is essentially a chromatography problem, the elution volume would impact the elution profile. So my comment was mostly regarding optimizing the elution profile to dial in the fraction collection.

Now if you only use gravity I personally would use batch elution and see if a single step elution might be of sufficient purity. I.e. once the gradient tells me when my target of interest starts eluting I would wash with a lower concentration to get rid of weakly bound proteins and then elute with the higher. Gradients are really only needed when there are other molecules with very similar affinities as the target and at least I did not have much luck with those using gravity columns. Recommended values I have seen are more in the area of 5-10 column volumes.