Purifying a target protein is an important step before investigating its structure and function. To purify a target protein from a mixture of substances, column chromatography is a useful technique due to its convenience and cost. This article provides a guide to help you choose the right agarose resins for protein purification using column chromatography.

In this article:

What is Column Chromatography?

Column Chromatography Preparation

What Features are Important to Consider before Choosing Agarose Resins?

1.Protein Size

2.Specific Binding Affinity

References

What is Column Chromatography?

Column chromatography is a common technique, used for research in protein biochemistry. This method is useful to purify a protein based on their features, such as its size or specific binding affinity to a ligand.

For example, to separate small molecules from unwanted large molecules in your sample, choose size exclusion chromatography by using a column and a porous matrix.

how size exclusion chromatography works to separate out target proteins within a column using agarose beads/resins

Otherwise, to separate a protein using its specific binding affinity to a ligand, use agarose beads cross-linked to a ligand.

how affinity chromatography works to separate out target proteins based on ligands and protein affinity

To learn more about how column chromatography works, read GoldBio article below:

How Column Chromatography Works to Separate Proteins

Common Terms used in Column Chromatography

Affinity chromatography

A chromatography technique to separate molecules based on their interaction with a ligand cross-linked to a matrix.


Agarose resin/Agarose bead

A common matrix used in column chromatography.


Analyte

A substance to be separated during chromatography.


Column

A tube used in the protein purification.


Column chromatography

A chromatography technique involving the use of a column containing a matrix (such as agarose beads).


Eluate

A solution containing the target protein after elution.


Eluent

A solution entering the column.


Elute

The process of dissolving the target protein.


Elution

The process of collecting the target molecule in a collection tube by using an elution buffer.


Elution buffer

A buffer for dissolving the target protein.


Gas chromatography

A chromatography technique to separate molecules by using a gas in the mobile phase.


Ligand

A molecule with a specific affinity that forms a complex with another molecule to perform a biological function.


Matrix

A solid material used as a stationary phase in the column.


Mobile phase

A mixture of analyte (substance being separated) and solvent flowing through the column.


Separated molecules

Molecules or proteins of interest.


Size exclusion chromatography

A chromatography technique to separate molecules based on size.


Solvent

A buffer to dissolve the substance being separated.


Stationary phase

A solid and immobile phase in the column.


Target protein

Protein of interest or purified protein. This can also be the separated molecules.


Wash

The removal of unbound molecules or nonspecific proteins by using a buffer.



Column Chromatography Preparation

To perform column chromatography, in addition to your sample, you will need a column, a matrix, a solvent to dissolve your sample, a washing buffer, and an elution buffer.

After gathering your supplies, you’ll need to pack the column with a matrix, such as agarose beads or resins. Keep in mind that you must avoid getting bubbles when packing your column.

To learn how to pack your column, watch this video below:



What Features are Important to Consider before Choosing Agarose Resins?

1.Protein Size

To separate proteins by their size, choose plain or cross-linked agarose beads.

a. Plain Agarose Beads

Plain agarose beads are sufficient when using gravity techniques, but not for use in high-pressure separation techniques. Gravity techniques use gravity to move the mixture of your sample and solvent (or mobile phase), whereas high-pressure techniques uses compressed gas to push the mobile phase in the column.

b. Cross-Linked Agarose Beads

Cross-linked beads are physically and chemically more stable than the plain agarose beads. Cross-linked beads are high-density agarose beads, treated with a particular reagent to produce more cross-linked polysaccharide chains, to add the strength and stability to the matrix.

Remember, when performing chromatography, you’re pouring a solvent through the column. This can cause pressure that would otherwise collapse the matrix. Therefore, physical stability in this situation is critical. In addition, the matrix should not react with any of the solutions during the purification.

For both plain and cross-linked agarose resins, there are different percentages of agarose resins: 2%, 4%, 6% or 8%, allowing better separation of molecules based on their size. The pore sizes are smaller in the higher percentage of agarose beads, so it’s useful to separate small globular proteins. Likewise, the lower percentage of agarose resins is better to separate larger globular proteins.

hot to choose agarose beads based on technique and protein size for chromatography. Choosing which agarose beads or resin to use depends on your purification method and then the size of proteins. Larger protein sizes require a low agarose percentage while small proteins require a higher agarose percentage.


2.Specific Binding Affinity

Another consideration when choosing your beads is specific binding affinity. It is another technique to purify your protein of interest by binding it to a ligand.

Some examples of protein features and structures used for affinity chromatography:


a. Particular Groups of Amino Acids

If your protein is comprised of carboxy group amino acids, aminoethyl-agarose beads allow covalent binding of the amino groups with carboxy groups. However, choose glyoxal-agarose beads, if your protein is comprised of lysine amino group.


b. The Affinity Tags

Affinity tags are short peptide sequences linked to your recombinant protein. These short peptide sequences often encode for a protein used for purification, labeling, detection, or visualization. During protein purification, an affinity tag of a protein binds to a ligand linked to the agarose resin.

Two common affinity tags used for protein purification:

The GST-tag

The GST-tag is a short protein encoding an enzyme, gluthathione S-transferase. This enzyme binds to a glutathione. For a protein with the GST tag, choose glutathione agarose resin. Glutathione is a highly efficient affinity purification tool because its affinity for GST is in the submillimolar range.

The His-tag

The His-tagged proteins are proteins with a short sequence containing a string of histidine residues. The histidine tags have affinity for metal ions.

how his-tag protein purification works - sequences of histidine residues have an affinity for metal ions. His-tag protein purification takes advantage of metal ion affinity in order to isolate target proteins

Purify His-tagged proteins by choosing agarose beads containing metal ions to perform immobilized metal affinity chromatography (IMAC). IMAC is a method to purify a protein with an affinity for metal ions, such as Co2+, Ni2+, Cu2+, or Zn2+, immobilized on a matrix (Yang et al., 1998).

NTA vs. IDA Agarose Beads

The metal ions are fixed on cross-linked agarose beads by a chelating ligand, nitilotriacetic acid (NTA) or iminodiacetic acid (IDA). NTA is more stable than IDA because it contains four chelating sites. IDA only has three chelating sites.

GoldBio offers several products for this purpose, such as: nickel agarose beads, cobalt agarose beads, zinc agarose beads, and copper agarose beads. A high-density resin helps increase the affinity of the resin, whereas a low density increases the specificity of the resin.

To learn more about His-Tag affinity for metal ions, read GoldBio article below:

His-Tag Metal Affinity Cations: What’s the difference again?


c. Immunoglobulins

Bacterial proteins serve as useful ligands for purifying polyclonal or monoclonal immunoglobulin G (IgG). These bacterial proteins include protein A and protein G. Both protein A and protein G , can bind to human and mammalian IgG antibodies, so they are useful as a ligand. However, protein A and protein G may have different affinities for IgG subclasses from different species.

Protein A and protein G bind to the Fc region of the immunoglobulins. Protein A binds to IgGs from different mammalian species, whereas protein G is effective for isolating, purifying immunoglobulins from cell culture media or biological fluids.

protein a agarose bead, protein g agarose bead

For antibody purification and immunoprecipitation, you can choose GoldBio protein A agarose resin or protein G agarose resin.


d. Enzyme-Substrate Interactions

The specific interaction between enzymes and their substrates provides a good tool for cleaving your molecule into fragments. This approach to enhance the performance of antibodies uses enzymes immobilized on agarose beads.

For purifying your antibody, you can then use a column with protein A or a column with G agarose beads to collect your specific fragments.

Some examples of these resins are:

  • Ficin agarose resin: This resin contains ficin, which is a cysteine protease. Ficin cleaves mouse IgG1 in the hinge region. As a result of this cleavage, a F(ab’)2 fragment is formed.

how ficin agarose beads work - what are ficin agarose beads and what do they do

  • Papain agarose resin: The agarose beads are linked to papain. This enzyme is a cysteine protease cleaving IgG molecules near the hinge region to produce an Fc domain and two monovalent Fab domains.

how papain agarose beads and resins work - what are papain agarose beads and how do they work

  • Pepsin agarose resin: Pepsin fixed to the matrix breaks down IgG generating F(ab’)2 fragments and small Fc fragments.

how pepsin agarose beads work - what are pepsin agarose beads and resins and what do they do

  • Trypsin agarose resin: Trypsin immobilized to agarose beads is a serine protease, which cleaves peptide bonds on the carboxy side of s-aminoethyl cysteine, arginine, and lysine residues. It is useful for producing peptides easily identified by mass spectrometry.

e. Biotinylated Molecules

A bacterial protein called streptavidin interacts non-covalently to a small molecule biotin (vitamin H/vitamin B7), making this protein an important tool for many molecular biology applications. As an example, to purify biotinylated antibodies, streptavidin agarose resin is useful for gravity flow columns, spin columns, and fast protein liquid chromatography.

how to choose agarose bead - complete guide for choosing agarose beads for affinity chromatography


References

Berg, J. M., Tymoczko, J. L., & Lubert Stryer. (2013). The Purification of Proteins Is an Essential First Step in Understanding Their Function. Nih.Gov; W H Freeman. https://www.ncbi.nlm.nih.gov/books/NBK22410/

Block, H., Maertens, B., Spriestersbach, A., Brinker, N., Kubicek, J., Fabis, R., Labahn, J., & Schäfer, F. (2009, January 1). Chapter 27 Immobilized-Metal Affinity Chromatography (IMAC): A Review (R. R. Burgess & M. P. Deutscher, Eds.). ScienceDirect; Academic Press. https://www.sciencedirect.com/science/article/pii/...

Böing, A. N., van der Pol, E., Grootemaat, A. E., Coumans, F. A. W., Sturk, A., & Nieuwland, R. (2014). Single-step isolation of extracellular vesicles by size-exclusion chromatography. Journal of Extracellular Vesicles, 3(1), 23430. https://doi.org/10.3402/jev.v3.23430.

Chivers, C. E., Koner, A. L., Lowe, E. D., & Howarth, M. (2011). How the biotin–streptavidin interaction was made even stronger: investigation via crystallography and a chimaeric tetramer. Biochemical Journal, 435(Pt 1), 55–63. https://doi.org/10.1042/BJ20101593.

Coskun, O. (2016). Separation Techniques: CHROMATOGRAPHY. Northern Clinics of Istanbul. https://doi.org/10.14744/nci.2016.32757.

Enzyme Immobilization on Polymeric Resins. (2008, October 1). GEN - Genetic Engineering and Biotechnology News. https://www.genengnews.com/magazine/99/enzyme-immo...

Gustavsson, P.-E., & Larsson, P.-O. (1996). Superporous agarose, a new material for chromatography. Journal of Chromatography A, 734(2), 231–240. https://doi.org/10.1016/0021-9673(95)01304-0.

Mohamad, N. R., Marzuki, N. H. C., Buang, N. A., Huyop, F., & Wahab, R. A. (2015). An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes. Biotechnology & Biotechnological Equipment, 29(2), 205–220. https://doi.org/10.1080/13102818.2015.1008192.

Stellwagen, E. (1990, January 1). [25] Gel filtration (M. P. Deutscher, Ed.). ScienceDirect; Academic Press. https://www.sciencedirect.com/science/article/pii/007668799082027Y.

Yang, Q., Tomlinson, A. J., & Naylor, S. (1998, January 1). Chapter 3 - Sample Preparation (Z. Deyl, I. Mikšik, F. Tagliaro, & E. Tesařová, Eds.). ScienceDirect; Elsevier. https://www.sciencedirect.com/science/article/abs/pii/S0301477008603012.