Affinity chromatography

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. 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. 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. ICT detection is highly specific to the microbe causing an infection. 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. Typical Biological Interaction Used in Affinity Chromatography