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 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.
to separate a protein using its specific binding affinity to a ligand, use agarose
beads cross-linked to a ligand.
To learn more about how column chromatography works, read GoldBio article below:
Common Terms used in Column Chromatography
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?
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.
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 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-tagged proteins are proteins with a short sequence containing a string of histidine residues. The histidine tags have affinity for metal ions.
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:
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.
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.
- 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.
- Pepsin agarose resin: Pepsin fixed to the matrix breaks down IgG generating F(ab’)2 fragments and small Fc fragments.
- 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.
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