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Purify, purify and…purify more: tips for improving your protein purification capabilities – part 3

After doing our best to purify our protein at the affinity chromatography step, we might decide we want an additional step of purification, which in many cases involves either an Ion-exchange or size exclusion chromatography. In this part I will focus on Ion-exchange chromatography and some tips on how to maximize your time and effort to get the most of this system.

Ion-exchange chromatography (IEC) binding step

Ion exchange chromatography is a powerful technique that is used on many cases at the middle of the purification process. I have discussed a method how to improve peak resolution by repetitive Ion-Exchange chromatography and today I would like to discuss general consideration when working with ion exchange columns.

Unlike affinity chromatography, the elution in IEC is conducted via increase in ion strength or gradual change in pH that may affect your protein behavior (structure and/or function). Since binding of the sample to the column should be at minimum salt interference and maximum surface charge, usually there will be a requirement to lower the salt concentration. If the previous step was a His tag Affinity chromatography, it will save time if the elution step will contain a minimal amount of salt (e.g., ~50mM NaCl) so there will be no need to exchange buffer. If you’re wondering about the effect of the Imidazole in the solution, it depends on the type of IEC column used. If you’re using a cationic exchanger or working under pH 7 you have no need to exchange the imidazole simply because the fact that imidazole has a pKa of ~7 for its first nitrogen, thus at higher pH the ring will not be charged and will not interfere with the binding of the protein to the matrix.

The beauty about IEC is the fact it is a capture method: you can load as much volume as you want as long as you don’t overload the column with protein mass. This means you can start from 50ml or even 500ml diluted solution assuming  you load your sample though a buffer valve. One thing to remember, though, is that there are proteins which do not bind to the anionic or cationic (or both!) so make sure you start collection immediately upon injection. Even in case your protein was not bound to the column, the contaminants might bind to the column so it is important to step-elute the bound material and evaluate it against the flowthrough and the pre-injected sample.

IEC elution step

When considering the elution step, two primary factors should be evaluated: elution buffer composition and elution methodology. In regard to the elution buffer it is usually recommended to use high concentration of salt (1-2M). pH shifts is less common since proteins are sensitive to small changes to the buffer’s pH which might affect their structure. On the other hand, it should be considered that gradual increase in salt can shift the protein from one oligomeric form to another (i.e. monomer vs. dimer) depending on its inter-molecular surface characteristics. Exploring other salts than the common NaCl is recommended when the protein aggregates or not eluting from the column.

The most common elution methodology is gradual increase in eluting buffer concentration (commonly known as “B buffer” or %B). While requiring more time, this methodology is essentially “fail-proof” as long as the gradient is relatively shallow such as 50%/50 min or 50%/10 column volume (CV) at 1ml/min flow rate using a 5ml column. Essentially you’ll want to elute the protein across 10-20CV so you’ll have better separation among close peaks. The slow rate will generate a much more concentrated elution which can expedite downstream concentration step.

Usually it is recommended to do a pilot run on the IEC. Inject small amount of the sample so you won’t overload the column (depending on the amount at hand, try to aim at 5-10mg) and evaluate the separation profile established with the current elution buffer composition and gradient. It is essential to couple a SDS-PAGE analysis of selected peaks together with the chromatogram so you can evaluate the following parameters:

  • Peak separation
  • Purity of the selected fractions
  • Amount of injected sample – should you increase/decrease the amount?

At this point you can decide on keeping or changing the current settings.

When analyzing the peaks, you should remember that proteins can bind to the matrix through differently charged surfaces, which means that the elution profile of a 95% protein can generate several elution peaks. That’s why it is crucial to perform an SDS-PAGE analysis of the major peaks to verify whether a certain elution  contains the protein of interest or a contamination. You should also remember that in certain cases what looks like a monomer on the gel can well be a dimer or an oligomer which forms a new charged patch that doesn’t exist in the monomer state; in certain cases, like crystallography, sample heterogeneity at the oligomeric level can be fatal for successful crystallization effort.

Step or gradient?

Moving from the gradient methodology to the step elution is a bit more complicated than it seems, especially when trying to separate between closely related peaks. Lets take the following scenario on the right.

IEC Gradient elution

In this purification run the main peak is bordered by two relatively small peaks. This was done by using an anion exchange column; color codes are blue color stands for 280nm, red for 254mn and brown for the conductivity in mS. Even though I would not recommend a shift from gradient to step in this particular case, I will use it to explain the workflow to perform such a change. First you should determine the volume in which you will want to start the step elution according to the absorbance curve (in this case I chose the inflection point, red dash line). From this volume deduct the total volume of the column (in this case 5ml) + additional 10% overhead in case of measurement errors. An additional way to determine the exact volume is to use the “%concentration” curve and determine the difference in volume between the starting point of mixing buffer A and B and the point at which there is increase in conductivity.  I have done so manually (without the %concentration function) and established the volume at ~115ml (blue dashed line) afterwhich I have deduced the conductivity value at this specific volume. This will be the first elution step that will remove the preceding contaminant peak. The same can be done to separate the main peak from the late contaminant peak. Once you figure this out, repeat the test run with this step elution and see if it fits with your needs.

Next post will discuss size exclusion chromatography – see you then!

Have any ideas or tips? great! write a comment below


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