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. 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.
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. 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.
Affinity chromatography is the basis for immunochromatographic test ICT strips, which provide a rapid means of diagnosis in patient care. Using ICT, a technician can make a determination at a patient's bedside, without the need for a laboratory.
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. 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. 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.
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 settling, 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. 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.
Many different affinity media exist for a variety of possible uses. Amino acid media is used with a variety of serum proteins, proteins, peptides, and enzymes, as well as rRNA and dsDNA.
Protein a chromatography for antibody purification pdf free
Carbohydrate bonding is most often used with glycoproteins or any other carbohydrate-containing substance; carbohydrate is used with lectins, glycoproteins, or any other carbohydrate metabolite protein. Dye ligand media is nonspecific, but mimics biological substrates and proteins. Glutathione is useful for separation of GST tagged recombinant proteins.
Heparin 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 media are most commonly used to target free carboxyl groups and proteins.
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.
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. 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 become 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. 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.
Most monoclonal antibodies have been purified using affinity chromatography based on immunoglobulin -specific Protein A or Protein G , derived from bacteria. 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. Histidine tags have an affinity for nickel , cobalt , zinc , copper and iron 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-agarose.
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. 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.