Affinity chromatography is a method for separating biochemical mixtures based on very specific interactions between antigens and antibodies, enzymes and substrates, receptors and ligands, or proteins and nucleic acids. This is a type of chromatographic laboratory technique used to purify biological molecules in a mixture by exploiting molecular properties. Biological macromolecules, such as enzymes and other proteins, interact with other molecules with high specificity through several types of bonding and interaction. Such interactions include hydrogen bonding, ionic interactions, disulfide bridges, hydrophobic interactions, and more. The high selectivity of affinity chromatography is caused by allowing the desired molecule to interact with the stationary phase and bonded within the column to be separated from the unwanted material that will not interact and elute first. No longer needed molecules are washed with buffers while the desired protein is released in the presence of an eluting solvent (higher salt concentration). This process creates a competitive interaction between the desired protein and stationary stationary molecules, ultimately allowing highly purified proteins to be released.
Video Affinity chromatography
Usage
Affinity chromatography can be used to purify and concentrate substances from the mixture into a buffer solution, reduce the amount of unwanted substances in the mixture, identify the biological compounds that bind to a particular substance, purify and concentrate the enzyme solution. Attractive molecules can be immobilized through covalent bonds. This occurs through an insoluble matrix such as a chromatographic medium such as cellulose or polyacrylamide. When the medium is tied to an attractive protein, it becomes immobile.
Maps Affinity chromatography
Principles
In short, affinity chromatography exploits differences in the strength of interactions between different biomolecules in the mobile phase, and the stationary phase. The first stationary phase is loaded into a column with a mobile phase containing various biomolecules from DNA to protein (depending on purification experiments). Then, the two phases are allowed time to bind. The washer buffer is then poured through a column containing both bonded phases. The washer buffer eliminates non-target biomolecules by interrupting their weaker interactions with stationary phases. Biomolecular targets have a much higher affinity for the stationary phase, and remain bonded to the stationary phase, not washed away by the washer. An elution buffer is then poured through a column containing the remaining target biomolecules. Elution buffers interfere with the interaction between targeted biomolecules tied to stationary to a much greater degree than washing buffers, effectively eliminating target biomolecules. This purified solution contains the elution buffer and the target biomolecule, and is called elution.
The stationary phase is usually the gel matrix, often of agarose; linear sugar molecules derived from algae. To prevent steric or overlapping disturbances during the process of binding the target molecule to the ligand, the inhibitor containing the hydrocarbon chain is first attached to the agarose bead (solid support). The inhibitor with this hydrocarbon chain is commonly known as the spacer between the agarose bead and the target molecule.
Usually, the starting point is a group of rough and heterogeneous molecules in all cell extracts, such as cell lysates, growth medium or blood serum. Attractive molecules will have properties that are known and defined, and can be exploited during the affinity purification process. The process itself can be considered a trap, with target molecules trapped in solid or stationary phases or mediums. Other molecules in the mobile phase will not get stuck because they do not own this property. The stationary phase can then be removed from the mixture, washed and the target molecule released from the trap in a process known as dialysis. The desired molecule is eluted with certain substances after washing the unrelated molecules. Thus, this produces a highly purified material. The very specific elution of the desired macromolecule from the stationary phase is usually effected by the addition of the elution buffer of a gradient of the same type to the macromolecule and substituting it. Perhaps 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 proteins or nucleic acids from a crude mixture.
If the molecular weight, hydrophobic, charge, and other proteins are unknown, affinity chromatography can still be applied to this situation. An example of this situation is when trying to find an enzyme with a specific activity, where it is possible to construct an affinity column with an inherent ligand that is similar or identical to the substrate of choice. The preferred enzyme will be eluted from a mixture based on the strong interaction of the immobilized enzyme and substrate analog, which will be selectively carried out through the affinity column. Then, elution of the enzyme with the appropriate substrate can be performed.
Batch settings and columns
Binding to the solid phase can be achieved by column chromatography where the solid medium is packed into a column, the starting mixture is run through the column to allow the washing, washing arrangement to be carried through the column and the elution buffer is subsequently applied to the column and collected.. These steps are usually performed at ambient pressure. Alternatively, binding can be achieved by using a batch treatment, for example, by adding the starting mixture to the solid phase in the vessel, mixing, separating the solid phase, removing the liquid phase, washing, re-centrifuging, adding elution buffer, re-centrifuging and removing elute.
Sometimes the hybrid method is used in such a way that the binding is done by batch method, but the solid phase with the target molecular bond is packed into the column and the washing and elution are carried out on the column.
Ligands used in affinity chromatography are obtained from organic and inorganic sources. Examples of biological sources are serum proteins, lectins, and antibodies. Inorganic sources as moronic action, chelate metal and triazine dye.
The third method, extending the absorption of the bed, which combines the advantages of the two methods mentioned above, has also been developed. Solid phase particles are placed in columns where the liquid phase is pumped from the bottom and out at the top. Particle gravity ensures that the solid phase does not come out of the column with the liquid phase.
Affinity columns can be eluted by altering the concentration of salt, pH, pI, charge and ion strength directly or through gradients to complete the particles of interest.
Recently, settings using more than one column in series have been developed. The advantage compared to a single column setup is that the resin material can be fully charged, since non-binding products are directly forwarded to successive columns with fresh column material. This chromatographic process is known as periodic counter-current chromatography (PCC). The cost of resin per product amount produced can be drastically reduced. Since a single column can always be eluted and regenerated while other columns are loaded, two columns are sufficient to fully benefit. Additional columns may provide additional flexibility for elution and regeneration time, with additional equipment costs and resin costs.
Custom use
Affinity chromatography can be used in a number of applications, including purification of nucleic acids, protein purification from cell-free extracts, and purification of blood.
By using affinity chromatography, one can separate proteins that bind certain fragments of proteins that do not bind to specific fragments. Since this purification technique depends on the biological properties of the required protein, it is a useful technique and the protein can be purified many folds in one step.
Various affinity media
Many different affinity media exist for various possible uses. In short, they (general):
- Enabled/Disabled - Serves as a functional spacer, supports matrices, and removes toxic reagent handling.
- Amino Acids - Used with various serum proteins, proteins, peptides, and enzymes, as well as rRNA and dsDNA.
- Avidin Biotin - Used in the process of purifying biotin/avidin and its derivatives.
- Carbohydrate Bonding - Most commonly used with glycoproteins or other substances containing carbohydrates.
- Carbohydrates - Used with lectins, glycoproteins, or other carbohydrate metabolite proteins.
- Dye Ligand - This medium is not specific, but mimics the biological substrate and protein.
- Glutathione - Useful for separation of recombinant proteins characterized by GST.
- Heparin - This medium is a common affinity ligand, and is particularly useful for separating plasma coagulation proteins, along with nucleic acid enzymes and lipases.
- Hydrophobic Interactions - Most commonly used to target free carboxyl groups and proteins.
- Immunoaffinity - Detailed below, this method uses the high specificity of antigens and antibodies to separate.
- Immobilized Metal Affinity Chromatography - Further details below, this method uses the interaction between metal ions and proteins (usually specifically marked) to be separated.
- Nucleotides/Coenzymes - Works to separate dehydrogenases, kinases, and transaminases.
- Nucleic Acid - Works to trap mRNA, DNA, rRNA, and other nucleic/oligonucleotides.
- Protein A/G - This method is used to purify immunoglobulin.
- Specialization - Designed for a particular class or protein/coenzyme type, this type of media will only serve to separate a particular protein or coenzyme.
Immunoaffinity
Another use for this procedure is the purification of the antibody affinity of the blood serum. If the serum is known to contain antibodies to certain antigens (for example if the serum originates from an organism immunized against the antigen in question) then the serum may be used for the purification of the antigen affinity. This is also known as Immunoaffinity Chromatography. For example, if an organism is immunized against a GST fusion protein it will produce antibodies to the fusion protein, and possibly antibodies to the GST tag as well. The protein can then be covalently combined with solid support such as agarose and used as an affinity ligand in the purification of antibodies from the immune serum.
For the accuracy of GST proteins and GST fusion proteins each can be combined separately. Serum was initially allowed to bind to the GST affinity matrix. This will remove antibodies against GST part of the fusion protein. The serum is then separated from solid support and allowed to bind to the GST-fusion protein matrix. This allows any antibody that recognizes the antigen to be captured in solid support. The most commonly achieved antibody elution uses low pH buffers such as glycine pH 2.8. These eluates are collected into neutral tris buffers or phosphates, to neutralize the low pH elution buffer and stop the degradation of antibody activity. This is a good example because affinity purification is used to purify the initial GST fusion protein, to remove unwanted anti-GST antibodies from serum and to purify the target antibodies.
Monoclonal antibodies can also be selected to bind proteins with high specificity, in which proteins are released under fairly soft conditions. This could be useful for further research in the future.
A simplified strategy is often used to purify the antibodies produced against peptide antigens. When peptide antigens are produced synthetically, terminal cysteine ​​residues are added to N- or C-terminus peptides. This cysteine ​​residue contains a sulfhydryl functional group that allows easily conjugated peptides to the carrier proteins (eg, Keyhole limpet hemocyanin (KLH)). The peptide containing the same cysteine ​​is also immobilized to the agarose resin through the cysteine ​​residue and then used to purify the antibody.
Most monoclonal antibodies have been purified using affinity chromatography based on Protein A or Protein G-specific immunoglobulins, derived from bacteria.
Chromatography affinity of metal immobilizing ions
Immobilized metal ion affinity chromatography (IMAC) is based on the specific coordination of amino acid covalent bonds, especially histidine, to metals. This technique works by enabling proteins with the affinity of metal ions to be stored in columns containing immobilized metal ions, such as cobalt, nickel, copper for the purification of proteins or peptides containing histidine, iron, zinc or gallium for the purification of phosphorylated proteins or peptides. Many natural proteins have no affinity for metal ions, so recombinant DNA technology can be used to introduce a protein tag into the relevant genes. The methods used to elucidate interesting proteins include altering pH, â € <â €
Perhaps the most common use of affinity chromatography is for the purification of recombinant proteins. Protein with known affinity are proteins that are marked to aid their purification. The protein may have been genetically modified to allow it to be selected for affinity binding; this is known as fusion protein. Tags include glutathione-S-transferase (GST), hexahistidine (Nya), and maltotic binding protein (MBP). Histidine tags have an affinity for nickel or cobalt ion that has been immobilized by forming coordinate coordinate coordinates with chelator incorporated in the stationary phase. For elution, a number of excess compounds that may act as metal ion ligands, such as imidazole, are used. GST has an affinity for glutathione that is commercially available as agarose glutathione. During elution, excess glutathione is used to replace marked proteins.
lectin
Lectin affinity chromatography is a form of affinity chromatography in which lectins are used to separate components in a sample. Lectins, such as concanavalin A, are proteins that can bind molecules of alpha-D-mannose carbohydrates and specific alpha-D-glucose. Some common carbohydrate molecules used in lectin affinity chromatography are Con A-Sepharose and WGA-agraose. Another example of lectin is a bran-agglutinin that binds D-N-acetyl-glucosamine. The most common application is to separate the glycoproteins from non-glycosylated proteins, or one glyoform from another glycopoform. Although there are various ways to perform affinity chromatography lectins, the goal is to extract the sugar ligand from the desired protein.
Custom
Another use for affinity chromatography is the purification of specific proteins using a gel matrix unique to a particular protein. For example, E. coli purification? -galactosidase is performed by affinity chromatography using p-aminobenyl-1-tio -? - D-galactopyranosyl agarose as an affinity matrix. p-aminobenyl-1-thio -? - D-galactopyranosyl agarose is used as an affinity matrix because it contains the 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 elute by adding the increased salt concentration to the column.
Alkaline Phosphatase from E. coli can be purified using the DEAE-Cellulose matrix. A. phosphatase has a slight negative charge, allowing it to bind weakly to the group of positively charged amines in the matrix. The enzyme can then be eluted out by adding a buffer with a higher salt concentration.
The affinity chromatography of boronate comprises the use of boronic acid or boronate to elute and calculate the amount of glycoprotein. Clinical adaptation has applied this type of chromatography to be used in determining the long-term assessment of diabetic patients through their glychemhemlobin analysis.
Serum albumin purification
Of the many uses of affinity chromatography, one of its uses is seen in the purification of the affinity of albumin and macroglobulin contamination. This type of purification is helpful in removing excess albumin and? 2 -macroglobulin contamination, when performing mass spectrometry. In the purification of serum albumin affinity, the stationary used to collect or attract serum proteins can be Cibacron Blue-Sepharose. Then the serum protein can be eluted from the adsorbent with a buffer containing thiocyanate (SCN - ).
Weak affinity chromatography
Weak affinity chromatography (WAC) is an affinity chromatography technique for affinity screening in drug development. WAC is an affinity-based liquid chromatography technique that separates chemical compounds based on different weak affinity for immovable targets. The higher the affinity of a compound to the target, the longer it remains in the separation unit, and this will be expressed as a longer retention time. The size of the affinity and affinity rating can be achieved by processing the retention time obtained from the compound being analyzed.
WAC technology is demonstrated against a number of different protein targets - protease, kinase, chaperone and target protein (PPI) interactions. WAC has proven to be more effective than the methods set for fragment-based screening.
References
External links
Source of the article : Wikipedia
0 Comments