Chromatography is the science of separation and we utilize it to isolate and purify proteins based on their unique physiochemical properties. One of the most fundamental and important skill sets a budding life scientist can master is protein chromatography.
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Ion-exchange chromatography is just one of many separation techniques used to purify proteins [1] and in this article, we will cover its basic principles, applications, and how to optimize this important method.
Highly pure proteins are vital for successful experiments. They play roles in research as assay reagents (for example, surface plasmon resonance), therapeutic candidates, and of course, as the subjects of structural and biochemical studies.
So, how does ion-exchange chromatography separate proteins?
Ion-exchange chromatography (IEX) separates proteins (or any biomolecules) based on differences in their net charge at a particular pH. Protein charge depends on the number and type of ionizable amino acid side chain groups. Each protein has an isoelectric point (pI), the pH at which the overall number of negative and positive charges is zero.
There are two fundamental concepts to understand before performing IEX. These are:
In principle, a protein could bind to either a cation or anion exchange resin, but in practice, proteins are only stable within a narrow pH range and the choice of the resin depends on the stability of the protein at a given pH.
Ion-exchange resins have charged functional groups bound to resin beads that attract biomolecules of the opposite charge. Cation exchange resins are negatively charged, and anion exchange resins are positively charged.
Resins are also categorized as &#;weak&#; or &#;strong&#; exchangers. These terms aren&#;t related to the strength of ion binding, but instead, refer to the extent that the ionization state of the functional groups varies with pH.
A &#;weak&#; exchanger is ionized over only a limited pH range, while a &#;strong&#; exchanger shows no variation in ion exchange capacity with changes in pH.
Weak exchange resins can gain or lose protons with changes in buffer pH, and that added variation in charge offers an additional dimension of selectivity for binding and elution.
Strong exchangers do not vary and remain fully charged over a broad pH range, which can make optimizing your separation simpler than with weak exchangers. Table 1 below summarizes the most common ion-exchange chromatography resins.
Table 1. Summary of ion-exchange chromatography resins and their properties.
Resin Abbreviation
Functional Group
Weak or Strong
Functional pH Range
DEAE
Diethylaminoethyl
[-N+(C2H5)2H+]
Weak anion
pH 2 - 9
ANX
Diethylaminopropyl
[-N+(C2H5)2H+]
Weak anion
pH 2 - 9
Q
Quaternary amine
[-N+(CH3)3]
Strong anion
pH 1 - 14
CM
Carboxymethyl
[-O-CH2-COO-]
Weak cation
pH 5 - 10
S
Methyl sulfonate
[O-CH2-CHOH-CH2-O-CH2-CHOH-CH2-SO3-]
Strong cation
pH 2 - 12
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SP
Sulfonyl
[-CH2-CH2-CH2-SO3-]
Strong cation
pH 2 -14
In addition to the type of functional group, you need to consider the physical properties of the resin. The size, material, and porosity of the resin beads dictate the maximum operating pressures and flow rates (which affect the speed of purification).
More importantly, bead size and porosity affect the resolution of the separation.
Larger beads are generally conducive to fast flow rates and provide resolution appropriate for early and intermediate stages of purification. Smaller beads provide the best resolution and are ideal for later-stage purification steps when purity is paramount.
The first step in designing an ion-exchange purification scheme should be the in silico determination of the pI of your protein of interest. The pI of a protein is determined by the aggregate charge of every amino acid in the protein chain.
The Henderson-Hasselbach equation is used to iteratively compute protein charge at certain pHs until one is found that produces a net protein charge of zero. The math can get complicated for proteins, but luckily, there are several online tools you can use to painlessly estimate your protein&#;s pI to guide your experiments. For example:
ProtParam, hosted by ExPASy, is the &#;classic&#; tool that most life scientists know of. You can calculate the theoretical molecular weight, isoelectric point, extinction coefficient, and other physiochemical properties based on your target protein sequence. The algorithm is based on the work of Bjellqvist et al. in the early s. [2,3]
Isoelectric Point Calculator (IPC) is a &#;new school&#; online tool. It computes a series of pI predictions using several published algorithms and pK datasets. In addition to displaying the range of computed isoelectric points, it also provides an average pI based on all methods. I find the output from IPC to be more useful and comprehensive than the value determined by ProtParam.
Proteome-pI is a database from which you can retrieve the pI of eukaryotic proteins. Calculating the pI of eukaryotic proteins is a little tricky because post-translational modifications (PTMs) can have a significant effect on your target protein&#;s pI.
The majority of PTMs occur on ionizable sidechains, and some PTMs, such as phosphorylation or acetylation, introduce new ionizable chemical groups.
So, if you are purifying a eukaryotic protein that is likely to be post-translationally modified, use the Proteome-pI tool to retrieve its pI.
Remember that the theoretical pI is likely to be different from the true isoelectric point, and may not reflect the actual charge distribution on the protein surface. The charge distribution is usually not uniform and a protein is capable of having both positively and negatively charged patches on its surface.
When working with a new protein, I always screen a range of cation and anion exchange resins, both weak and strong, to optimize this purification step.
It&#;s important to tailor your ion-exchange chromatography experiment to your target protein to ensure the best outcome: maximum purity. There are loads of parameters you can optimize to achieve this, including:
The choice of a buffer system, its pH, additives, and salt concentration all have a direct effect on the success of your ion-exchange chromatography experiment. Buffer scouting is frequently required to find the optimal pH for solubility and adsorption of your protein sample to the ion-exchange chromatography resin.
When screening resins and buffer conditions, keep the following in mind:
Proteins are most often eluted from ion-exchange chromatography columns by increasing the concentration of counterions (salts) in the buffer solution. You may also consider using pH shifts as well which can be helpful in specific cases when using a weak ion-exchange resin. Or when adjusting the salt concentration cannot achieve sufficient resolution.
Choice of elution method, either linear gradient or a step elution, affects selectivity. Also, keep in mind that downstream techniques may be complicated by high salt concentrations or elution buffer pHs.
Linear gradients gradually raise the ionic strength and are ideal when starting with an unknown sample or if peak resolution is important. Peak resolution is also improved by reducing flow rates, eluting over a greater volume, or eluting with a shallower gradient (a smaller increase in salt concentration or pH per unit volume of elution buffer).
Step elution speeds up the purification process and minimizes the final protein elution volume, however, it provides poor resolution and should be used once the IEX separation has been optimized.
You could combine these two approaches and include a high-stringency wash step, and then start a linear gradient at a higher concentration of salt to elute your sample.
Differential column chromatography (sometimes referred to as group elution or flow-through mode) is used to remove contaminants by choosing conditions that maximize binding of the contaminants and allow target proteins to pass through the column.
It&#;s especially helpful in removing contaminants and improving column specificity in a later purification step.
It&#;s also a popular and effective strategy for removing nucleic acid contaminants because DNA and RNA are highly negatively charged at a neutral-to-basic pH. So, they bind strongly to anion exchange resins but not to cation exchange resins.
Ion-exchange chromatography is an incredibly versatile method for protein purification, which is critical to certain experiments such as surface plasmon resonance and structural biology. It may be used at any stage of purification, and the diversity of available resins provide a broad spectrum of selectivity that can be fine-tuned to your protein of interest.
Has this article helped you to understand ion-exchange chromatography? Has it enabled you to optimize your ion-exchange experiment? If so, please comment below and share what kind of snafus you have run into.
Originally published January . Reviewed and republished on October .
Ion exchange (IEX) chromatography is a technique that is commonly used in biomolecule purification. It involves the separation of molecules on the basis of their charge.
This technique exploits the interaction between charged molecules in a sample and oppositely charged moieties in the stationery phase of the chromatography matrix. This type of separation is difficult using other techniques as charge is easily manipulated by the pH of buffer used.
Two types of ion exchange separation is possible - cation exchange and anion exchange. In anion exchange the stationary phase is positively charged whilst in cation exchange it is negatively charged.
IEX chromatography is used in the separation of charged biomolecules. The crude sample containing charged molecules is used as the liquid phase. When it passes through the chromatographic column, molecules bind to oppositely charged sites in the stationary phase.
The molecules separated on the basis of their charge are eluted using a solution of varying ionic strength. By passing such a solution through the column, highly selective separation of molecules according to their different charges takes place.
Key steps in the ion exchange chromatography procedure are listed below:
A pH gradient can also be applied to elute individual proteins on the basis of their isoelectric point (pI) i.e. the point at which the amino acids in a protein carry neutral charge and hence do not migrate in an electric field. As amino acids are zwitter ionic compounds they contain groups having both positive and negative charges. Based on the pH of the environment, proteins carry a positive, negative, or nil charge. At their isoelectric point, they will not interact with the charged moieties in the column resin and hence are eluted. A decreasing pH gradient can be used to elute proteins using an anion exchange resin and an increasing pH gradient can be used to elute proteins from cation exchange resins. This is because increasing the buffer pH of the mobile phase causes the protein to become less protonated (less positively charged) so it cannot form an ionic interaction with the negatively charged resin, allowing is elution. Conversely, lowering the pH of the mobile phase will cause the molecule to become more protonated (less negatively charged_, allowing its elution.
Ion exchange resins have positively or negatively charged functional groups covalently linked to a solid matrix. Matrices are usually made of cellulose, polystyrene, agarose, and polyacrylamide. Some of the factors affecting resin choice are anion or cation exchanger, flow rate, weak or strong ion exchanger, particle size of the resin, and binding capacity. The stability of the protein of interest dictates the selection of an anion or a cation exchanger &#; either exchanger may be used if the stability is of no concern.
Ion exchange is the most widely used chromatographic method for the separation and purification of charged biomolecules such as polypeptides, proteins, polynucleotides, and nucleic acids. Its widespread applicability, high capacity and simplicity, and its high resolution are the key reasons for its success as a separation method. Ion exchange chromatography is widely used in several industrial applications some of which are as follows:
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