I now work in a lab with a protein and a pre-made purification protocol, were they purify a His-tagged protein using linear elution which then gives a steady increase in the imidazole concentration. However, I am curious to as if you plainly have to test which elution scheme you want to use, or if there are some "rule-of thumb" on what is a more standard way to elute protein, and what in general is more common?

Also, say for instance (just as an example) that I use a protocol which in the end loses about 80% of the protein before it's pure, is one of the first things you could do is to change the elution schemes to increase the yield? Are there other very basic things one can do to optimize a purification protocol which has not been so thoroughly optimized?

So basically:

  1. Is linear "usually" better/more common than stepwise?
  2. Is imidazole gradient "usually" better/more common than pH gradient?

Linear is better typically. The flow rate combined with fraction size and length of time gives you better resolution for which proteins are coming off the column at what point.

Once you've narrowed down the imidazole concentration at which your protein comes off the column, you can in future just stick to concentrations around that area, though it's still advisable to use gradients either side of that concentration as your preps are never likely to be completely consistent.

How do you know you're losing 80% of your protein before achieving purity? If your protein isn't separating from other contaminants during IMAC, you can try switching to an IMAC optimised strain (I use NEB BL21(DE3) NiCo21's).

Usually you'll have to do some downstream purification in any event. Gel filtration is common. You could also consider a different column chemistry such as anion exchange.


Typically I wouldn't expect that you would need to mess with pH very much when performing IMAC. I certainly have never had to. You are likely to need physiological pH to ensure native folding of your protein.

You can however use other additives if your protein is recalcitrant. For example, I often add 1mM DTT and 2M urea to my samples and then heat them slightly as my protein binds things easily and is extremely stable. GE Provide a whole booklet of supported additives for their columns, you may have the same thing for yours too.

  • $\begingroup$ thanks for the input, the optimization is just because I'm curios in general what steps could easily be altered a bit and most likely give you metter protein yield. I just stated 80% as an example, but that might be a bit extreme. Although I am working on a protocol that i know they really just went with without really trying to optimize it. $\endgroup$ – CuriousTree Jan 14 '17 at 17:05
  • $\begingroup$ You can't say categorically that linear is better. There are cases where increasing the imidazole concentration step-wise results in cleaner, more concentrated fractions, and other cases where steadily increasing its concentration via a gradient over a period of time works better. It all depends on the protein and the starting material. $\endgroup$ – MattDMo Jan 14 '17 at 19:47
  • $\begingroup$ For the purposes of calibration and optimisation linear is typically better so I stand by my point. Stepwise doesn't give you the same resolution. Once you've established your ballpark elution concentration then sure. $\endgroup$ – Joe Healey Jan 14 '17 at 19:50
  • $\begingroup$ This does answer what I'm wondering about (thanks), however what about pH vs. imidazole concentration? $\endgroup$ – CuriousTree Jan 15 '17 at 9:55
  • $\begingroup$ Edited my answer $\endgroup$ – Joe Healey Jan 15 '17 at 10:00

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