Affinity chromatography is a method of separating biochemical mixture based on a highly specific interaction between antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid. It is a type of chromatographic laboratory technique used for purifying biological molecules within a mixture by exploiting molecular properties. Biological macromolecules, such as enzymes and other proteins, interact with other molecules with high specificity through several different types of bonds and interaction. Such interactions include hydrogen bonding, ionic interaction, disulfide bridges, hydrophobic interaction, and more. The high selectivity of affinity chromatography is caused by allowing the desired molecule to interact with the stationary phase and be bound within the column in order to be separated from the undesired material which will not interact and elute first. The molecules no longer needed are first washed away with a buffer while the desired proteins are let go in the presence of the eluting solvent (of higher salt concentration). This process creates a competitive interaction between the desired protein and the immobilized stationary molecules, which eventually lets the now highly purified proteins be released.
- 1 Uses
- 2 Principle
- 3 Batch and column setups
- 4 Specific uses
- 5 Weak affinity chromatography
- 6 References
- 7 External links
Affinity chromatography can be used to purify and concentrate a substance from a mixture into a buffering solution, reduce the amount of unwanted substances in a mixture, identify the biological compounds binding to a particular substance, purify and concentrate an enzyme solution. The molecule of interest can be immobilized through covalent bonds. This occurs through an insoluble matrix such as chromatographic medium like cellulose or polyacrylamide. When the medium is bound to the protein of interest it becomes immobilized.
In summary, affinity chromatography exploits the differences in interactions' strengths between the different biomolecules within a mobile phase, and the stationary phase. The stationary phase is first loaded into a column with mobile phase containing a variety of biomolecules from DNA to proteins (depending on the purification experiment). Then, the two phases are allowed time to bind. A wash buffer is then poured through a column containing both bound phases. The wash buffer removes non-target biomolecules by disrupting their weaker interactions with the stationary phase. Target biomolecules have a much higher affinity for the stationary phase, and remain bound to the stationary phase, not being washed away by wash buffer. An elution buffer is then poured through the column containing the remaining target biomolecules. The elution buffer disrupts interactions between the bound target biomolecules with the stationary to a much greater extent than the wash buffer, effectively removing the target biomolecules. This purified solution contains elution buffer and target biomolecules, and is called elution.
The stationary phase is typically a gel matrix, often of agarose; a linear sugar molecule derived from algae. To prevent steric interference or overlap during the binding process of the target molecule to the ligand, an inhibitor containing a hydrocarbon chain is first attached to the agarose bead (solid support). This inhibitor with a hydrocarbon chain is commonly known as the spacer between the agarose bead and the target molecule.
Usually, the starting point is a crude, heterogeneous group of molecules in a whole cell extract, such as a cell lysate, growth medium or blood serum. The molecule of interest will have a well known and defined property, and can be exploited during the affinity purification process. The process itself can be thought of as an entrapment, with the target molecule becoming trapped on a solid or stationary phase or medium. The other molecules in the mobile phase will not become trapped as they do not possess this property. The stationary phase can then be removed from the mixture, washed and the target molecule released from the entrapment in a process known as dialysis. The desired molecules are eluted with specific substances after washing the non-interacting molecules away. Thus, this results in a highly purified material. Highly specific elution of the desired macromolecule from the stationary phase is usually effected by adding to the eluting buffer a gradient of the same kind on the macromolecule and displaces it. Possibly the most common use of affinity chromatography is for the purification of recombinant proteins. Affinity chromatography is an excellent choice for the first step in purifying a protein or nucleic acid from a crude mixture.
If the molecular weight, hydrophobicity, charge, etc. of a protein is unknown, affinity chromatography can still apply to this situation. An example of this situation is when trying to find an enzyme with a particular activity, where it can be possible to build an affinity column with an attached ligand that is similar or identical to the substrate of choice. The way that the desired enzyme would be eluted would be from the mixture based on the strong interaction of enzyme and the immobilized substrate analog, which would be done selectively through the affinity column. Then, the elution of the enzyme with the appropriate substrate can be done.
Batch and column setups
Binding to the solid phase may be achieved by column chromatography whereby the solid medium is packed onto a column, the initial mixture run through the column to allow setting, a wash buffer run through the column and the elution buffer subsequently applied to the column and collected. These steps are usually done at ambient pressure. Alternatively, binding may be achieved using a batch treatment, for example, by adding the initial mixture to the solid phase in a vessel, mixing, separating the solid phase, removing the liquid phase, washing, re-centrifuging, adding the elution buffer, re-centrifuging and removing the elute.
Sometimes a hybrid method is employed such that the binding is done by the batch method, but the solid phase with the target molecule bound is packed onto a column and washing and elution are done on the column.
The ligands used in affinity chromatography are obtained from both organic and inorganic sources. Examples of biological sources are serum proteins, lectins and antibodies. Inorganic sources as moronic acts, metal chelates and triazine dyes.
A third method, expanded bed absorption, which combines the advantages of the two methods mentioned above, has also been developed. The solid phase particles are placed in a column where liquid phase is pumped in from the bottom and exits at the top. The gravity of the particles ensure that the solid phase does not exit the column with the liquid phase.
Affinity columns can be eluted by changing salt concentrations, pH, pI, charge and ionic strength directly or through a gradient to resolve the particles of interest.
More recently, setups employing more than one column in series have been developed. The advantage compared to single column setups is that the resin material can be fully loaded, since non-binding product is directly passed on to a consecutive column with fresh column material. These chromatographic processes are known as periodic counter-current chromatography (PCC). The resin costs per amount of produced product can thus be drastically reduced. Since one column can always be eluted and regenerated while the other column is loaded, already two columns are sufficient to make full use of the advantages. Additional columns can give additional flexibility for elution and regeneration times, at the cost of additional equipment and resin costs.
Affinity chromatography can be used in a number of applications, including nucleic acid purification, protein purification from cell free extracts, and purification from blood.
By using affinity chromatography, one can separate proteins that bind a certain fragment from proteins that do not bind that specific fragment. Because this technique of purification relies on the biological properties of the protein needed, it is a useful technique and proteins can be purified many folds in one step.
Various affinity media
Many different affinity media exist for a variety of possible uses. Briefly, they are (generalized):
- Activated/Functionalized – Works as a functional spacer, support matrix, and eliminates handling of toxic reagents.
- Amino Acid – Used with a variety of serum proteins, proteins, peptides, and enzymes, as well as rRNA and dsDNA.
- Avidin Biotin – Used in the purification process of biotin/avidin and their derivatives.
- Carbohydrate Bonding – Most often used with glycoproteins or any other carbohydrate-containing substance.
- Carbohydrate – Used with lectins, glycoproteins, or any other carbohydrate metabolite protein.
- Dye Ligand – This media is nonspecific, but mimics biological substrates and proteins.
- Glutathione – Useful for separation of GST tagged recombinant proteins.
- Heparin – This media is a generalized affinity ligand, and it is most useful for separation of plasma coagulation proteins, along with nucleic acid enzymes and lipases.
- Hydrophobic Interaction – Most commonly used to target free carboxyl groups and proteins.
- Immunoaffinity – Detailed below, this method utilizes antigens' and antibodies' high specificity to separate.
- Immobilized Metal Affinity Chromatography – Detailed further below, this method uses interactions between metal ions and proteins (usually specially tagged) to separate.
- Nucleotide/Coenzyme – Works to separate dehydrogenases, kinases, and transaminases.
- Nucleic Acid – Functions to trap mRNA, DNA, rRNA, and other nucleic acids/oligonucleotides.
- Protein A/G – This method is used to purify immunoglobulins.
- Speciality – Designed for a specific class or type of protein/co enzyme, this type of media will only work to separate a specific protein or coenzyme.
Another use for the procedure is the affinity purification of antibodies from blood serum. If the serum is known to contain antibodies against a specific antigen (for example if the serum comes from an organism immunized against the antigen concerned) then it can be used for the affinity purification of that antigen. This is also known as Immunoaffinity Chromatography. For example, if an organism is immunised against a GST-fusion protein it will produce antibodies against the fusion-protein, and possibly antibodies against the GST tag as well. The protein can then be covalently coupled to a solid support such as agarose and used as an affinity ligand in purifications of antibody from immune serum.
For thoroughness the GST protein and the GST-fusion protein can each be coupled separately. The serum is initially allowed to bind to the GST affinity matrix. This will remove antibodies against the GST part of the fusion protein. The serum is then separated from the solid support and allowed to bind to the GST-fusion protein matrix. This allows any antibodies that recognize the antigen to be captured on the solid support. Elution of the antibodies of interest is most often achieved using a low pH buffer such as glycine pH 2.8. The eluate is collected into a neutral tris or phosphate buffer, to neutralize the low pH elution buffer and halt any degradation of the antibody's activity. This is a nice example as affinity purification is used to purify the initial GST-fusion protein, to remove the undesirable anti-GST antibodies from the serum and to purify the target antibody.
Monoclonal antibodies can also be selected to bind proteins with great specificity, where protein is released under fairly gentle conditions. This can be come of use for further research in the future.
A simplified strategy is often employed to purify antibodies generated against peptide antigens. When the peptide antigens are produced synthetically, a terminal cysteine residue is added at either the N- or C-terminus of the peptide. This cysteine residue contains a sulfhydryl functional group which allows the peptide to be easily conjugated to a carrier protein (e.g. Keyhole limpet hemocyanin (KLH)). The same cysteine-containing peptide is also immobilized onto an agarose resin through the cysteine residue and is then used to purify the antibody.
Immobilized metal ion affinity chromatography
Immobilized metal ion affinity chromatography (IMAC) is based on the specific coordinate covalent bond of amino acids, particularly histidine, to metals. This technique works by allowing proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions, such as cobalt, nickel, copper for the purification of histidine-containing proteins or peptides, iron, zinc or gallium for the purification of phosphorylated proteins or peptides. Many naturally occurring proteins do not have an affinity for metal ions, therefore recombinant DNA technology can be used to introduce such a protein tag into the relevant gene. Methods used to elute the protein of interest include changing the pH, or adding a competitive molecule, such as imidazole.
Possibly the most common use of affinity chromatography is for the purification of recombinant proteins. Proteins with a known affinity are protein tagged in order to aid their purification. The protein may have been genetically modified so as to allow it to be selected for affinity binding; this is known as a fusion protein. Tags include glutathione-S-transferase (GST), hexahistidine (His), and maltose binding protein (MBP). Histidine tags have an affinity for nickel or cobalt ions which have been immobilized by forming coordinate covalent bonds with a chelator incorporated in the stationary phase. For elution, an excess amount of a compound able to act as a metal ion ligand, such as imidazole, is used. GST has an affinity for glutathione which is commercially available immobilized as glutathione agarose. During elution, excess glutathione is used to displace the tagged protein.
Lectin affinity chromatography is a form of affinity chromatography where lectins are used to separate components within the sample. Lectins, such as concanavalin A are proteins which can bind specific alpha-D-mannose and alpha-D-glucose carbohydrate molecules. Some common carbohydrate molecules that is used in lectin affinity chromatography are Con A-Sepharose and WGA-agraose. Another example of a lectin is wheat germ agglutinin which binds D-N-acetyl-glucosamine. The most common application is to separate glycoproteins from non-glycosylated proteins, or one glycoform from another glycoform. Although there are various ways to perform lectin affinity chromatography, the goal is extract a sugar ligand of the desired protein.
Another use for affinity chromatography is the purification of specific proteins using a gel matrix that is unique to a specific protein. For example, the purification of E. coli β-galactosidase is accomplished by affinity chromatography using p-aminobenyl-1-thio-β-D-galactopyranosyl agarose as the affinity matrix. p-aminobenyl-1-thio-β-D-galactopyranosyl agarose is used as the affinity matrix because it contains a galactopyranosyl group, which serves as a good substrate analog for E.Coli-B-Galactosidase. This property allows the enzyme to bind to the stationary phase of the affinity matrix and is eluted by adding increasing concentrations of salt to the column.
Alkaline phosphatase from E. coli can be purified using a DEAE-Cellulose matrix. A. phosphatase has a slight negative charge, allowing it to weakly bind to the positively charged amine groups in the matrix. The enzyme can then be eluted out by adding buffer with higher salt concentrations.
Boronate affinity chromatography
Boronate affinity chromatography consists of using boronic acid or boronates to elute and quantify amounts of glycoproteins. Clinical adaptations have applied this type of chromatography for use in determining long term assessment of diabetic patients through analysis of their glycated hemoglobin.
Serum albumin purification
Of many uses of affinity chromatography, one use of it is seen in affinity purification of albumin and macroglobulin contamination. This type of purification is helpful in removing excess albumin and α2-macroglobulin contamination, when performing mass spectrometry. In affinity purification of serum albumin, the stationary used for collecting or attracting serum proteins can be Cibacron Blue-Sepharose. Then the serum proteins can be eluted from the adsorbent with a buffer containing thiocyanate (SCN−).
Weak affinity chromatography
Weak affinity chromatography (WAC) is a affinity chromatography technique for affinity screening in drug development. WAC is an affinity-based liquid chromatographic technique that separates chemical compounds based on their different weak affinities to an immobilized target. The higher affinity a compound has towards the target, the longer it remains in the separation unit, and this will be expressed as a longer retention time. The affinity measure and ranking of affinity can be achieved by processing the obtained retention times of analyzed compounds.
The WAC technology is demonstrated against a number of different protein targets – proteases, kinases, chaperones and protein–protein interaction (PPI) targets. WAC has been shown to be more effective than established methods for fragment based screening.
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