What is Affinity Chromatography?
The majority of affinity chromatography techniques are comprised of a stationary phase (solid phase) and the mobile phase. Mobile phase refers to your cells lysate, or any other mixture that is made up of biomolecules. A ligand which binds to the targeted molecule is covalently attached with the solid. The interactions between the solid and mobile phase is exploited through affinity chromatography to produce the desired substance in pure form.
The target molecule is bound to the ligand while the majority of other molecules pass through. The biomolecule of the target is degraded through changing conditions (pH or concentration of salt) or through the competition with a free ligand.The most crucial property the solid phase should possess is the ability to immobilize ligand. Many materials, such as acrylates and silica gels work well.
To stop steric interference of the target molecule with the ligand the inhibitor is bonded in the phase of solid. This inhibitor is referred to by the name of spacer. The most common spacer can be described as an inhibitor with an Hydrocarbon Chain (CH2 spacer). Chemicals such as cyanogen bromide and epoxy can functionize the solid phase using hydrocarbon chains that result in different lengths of the carbon chain according to the chemical.
Principle of Affinity chromatography
The basis of affinity chromatography is the fact that its stationary component is made up of a medium (e.g. cellulose beads) where the substrate (or often coenzyme) has been covalently bound and in such a way that the reactive elements that are necessary for binding to enzymes are exposed. When the mix of proteins is filtered across the chromatography columns the proteins that possess an affinity site that is immobilized will attach onto the stationary part and all otter proteins will be eliminated in the empty space that is the column.
After all the other proteins have been removed and bound, it is now possible to bind the enzyme(s) are able to be removed in a variety of ways:
- by increasing the ionic strength this buffer e.g. by introducing the sodium chloride gradient which weakens the interactions that the enzyme has with its immobilised substrate
- by altering by altering the pH in the buffer. This is thereby lessening the interaction with the substrate and enzyme
- by adding a significant amount of substrate (or an analogue of the substrate) to the buffer for elution, to ensure that there is a competing between the immobilised and free substrates for the enzyme protein
Linking the substrate to the support medium
- There are a variety of activated agarose gels which can be used to connect ligands CNBr-agarose is a simple choice to attach amines, however it does not have a large space between the beads of gel and bound ligand. This means that protein binding could be hindered by steric factors.
- Aminohexanoic acid-agarose (CH-agarose, for reaction with amines) and diaminohexane-agarose (AH-agarose, for reaction with carboxylic acids)) have relatively long methylene chains that keep the ligand a significant distance from the gel beads.
- An alternative reagent for attaching amines is carbonyldiimadazole-agarose, and epoxy-activated agarose is used for alcohols.
Components of affinity chromatography
The matrix acts as an inert structure that a ligand may be connected directly or indirectly. The most efficient matrix materials are polyacrylamide and agarose. It is distinguished by its unique properties like;
- Doesn’t itself absorb molecules in a substantial quantity.
- Ligands should be coupled without altering the binding properties.
- Stability in a variety of conditions for experiments, such as both low and high pH detergent, as well as dissociating conditions.
Materials utilized in Matrix;
|Agarose||hydrophilic, almost no unspecific bonds, the gold standard for protein purification|
|Silica gel||nanoporous (leads to unspecific bonds), functionalized via silane, silanes are washed away by alkaline buffer → reduced stability, applications: bound nucleic acids chaotropic|
|Aluminium oxide||acidic surface, binds amines irreversibly, used to reduce the amount of specific substances|
|Acrylate||partially hydrophobic (unspecific bounds possible), monodisperse particles, used for cell separation|
|Organic polymers||partially hydrophobic (unspecific bounds possible), monodisperse particlescan be used for ligand coupling, not recommended for protein purification because of unspecific bounds|
2. Spacer arm
It prevents the ligand from attaching to the matrix, which could interfere with its ability to bond to macromolecules. The optimal length is 6-10 carbon atoms or equivalent. Most commonly, it is utilized for smaller immobilized ligands. Examples of Spacer arms are 1,6-diamino Hexane and 6-amino Hexanoic Acid.
The most commonly used coupling systems include:
|Epoxide with C6 acid||C10|
The ligand is a molecule which binds reversibly with an individual chemical or group of molecules, making it possible to purify the sample using affinity chromatography. The choice of a specific ligand to be used for affinity chromatography can be influenced by two aspects:
- the ligand has to show a an irreversible and specific binding affinity for the targeted substance(s)
- It should possess chemically modifiable groups that permit it to be bonded to the matrix without damaging the binding function.
Any element can be utilized as a ligand for purification of the binding partner of its choice. A few of the most common biological interactions often used in affinity chromatography are as follows:
- Enzyme used for substrate analog and inhibitors, as well as cofactor.
- Antibody used for antigen, virus, cell.
- Lectin is a polysaccharide glycoprotein cell surface receptor cell.
- Nucleic acid is used to complement base sequences, histones nucleic acid polymerase and nucleic acid-binding protein.
- Hormone vitamin, used to receptors, carriers protein.
- Glutathione used for glutathione-S-transferase or GST fusion proteins.
- Metal ions that are used in Poly (His) fusion proteins and native proteins that contain histidine, cysteine or tryptophan residues on their surface.
Most commonly used ligands include;
|Chelator + Ni-, Co-ions||His-tagged proteins|
Steps of Affinity Chromatography
Step: 1 Attach ligand to column matrix
The binding of the ligand with the matrix requires that a covalent bond is created between both. This is accomplished through derivatization of the sugar-based”hydroxyl groups. It is essential to understand that the substrate may not be able to access the active site of the ligand, in the event that it is hidden inside the ligand. The majority of ligands are connected to spacer arms that are then attached onto the matrix. The matrix-ligand gel is loaded into an column of elution.
Step: 2 Load Protein Mixture onto the Column
After the column is made, the mix containing isolate is then poured into the column for elution. Gravity pulls the solution into the gel because most of the proteins don’t attach to the ligand-matrix. If ligand that is recognized as substrate moves through the gel it bonds to the ligand-matrix complex, stopping its flow within the gel. Certain impurities pass through the gel because of gravity, however the majority remain unbound in the gel column.
Step 3: Proteins Binds to the ligand
To remove these impurities which are not bound the wash must be of high pH or salt concentration or temperature is passed across the gel. It is crucial to make the most powerful wash possible to ensure all impurities are eliminated. After the impurities have been removed then the only thing left of the protein mixture must be the specific isolates.
Step 4: Wash the column to remove the unwanted Materials
To finally collect the an isolate that is attached to the ligand matrix in the gel second wash is run over the column.
Step 5: Wash off the Proteins that are loosely bind
The second wash is based on the reversible properties of binding of the ligand. This lets the bound protein separate from its ligand in the presence of the more powerful wash.
Step 6: Elute proteins that are tightly bound to ligand and collect purified protein and interest
The protein then has the freedom to pass through the gel before being removed.
Elution methods of Affinity Chromatography
1. pH elution
A variation in pH alters the amount of ionization by charged groups on the ligand as well as proteins bound. The alteration could affect site of binding directly decreasing its affinity or trigger an indirect change in affinity due to modifications in the conformation.
A gradual decrease by a step in pH can be the most popular method of eluting bound substances. Chemical stabilities of the matrix the ligand, and the target protein determines the limits of pH which can be used. If a low pH is required then you should collect the fractions into neutralization buffers like 1 M Tris-HCl pH 9 (60-200 mg ul for each ml fraction eluted) to bring the fraction back to an equilibrium pH. The column should be returned in the neutral pH immediately.
2. Ionic strength elution
The exact mechanism behind elimination through changes in the strength of ionic depends on the specific relationship between the ligand and the target protein. This is a moderate elimination using a buffer that has higher Ionic strength (usually NaCl), applied in a linear manner as well as in steps. Enzymes typically elute with a concentration of 1 million NaCl and less.
3. Competitive elution
They are typically used to distinguish substances in an individual medium or when the attraction of the target protein interactions is quite high. The eluting agent is competing with the protein of interest or for binding to the binding ligand. Substances can be eluted by the concentration gradient of one Eluent, or by pulse elimination.
In the case of competitive elution the concentration of the competing compound should be equal to that of the ligand that is coupled. If, however, the competing compound is able to bind more easily than the ligand targeted molecule, you should use an amount that is ten times greater than the ligand.
4. Reduced polarity of eluent
Conditions are employed to reduce the polarity of the eluent and allow for elution but not inactivating the substances that are eluted. Dioxane (up to 10 10%) as well as Ethylene glycol (up to 50 percent) are common to this kind of liquid eluent.
5. Chaotropic eluents
If other methods of elution fail the deforming buffers which alter the protein’s structure can be employed, e.g. Chaotropic agents like the guanidine hydrochloride and urea. Avoid chaotropes whenever possible as they can cause to cause denature of the protein eluted.
Types of Affinity Chromatography
1. Lectin Affinity Chromatography
- Purification of glycoproteins, specifically the membrane-receptor proteins.
- Lectins are a class of proteins made by animals and plants that are able to bind glycoproteins and carbohydrates.
- Useful to separate cells into different types, making use of saccharide component of their outer membranes.
- The most commonly used lectins include: ConcanavalinA Soyabean lectin, etc..
2. Immuno Affinity chromatography
- It is used in the isolation and elimination of a variety of proteins such as antigens, membrane proteins that are of viral origin.
- It is used to purify antibodies.
- Ligands used include the Protein A as well as protirn G.
3. Metal Chelate Chromatography
- Special type of chromatography which immobilised metal ions such as Cu2+ Zn2+ , Mn2+, Ni2+ etc. are employed.
- It is used to purify proteins that contain imidazole groups or indole groups.
- Commonly metal ions are immobilised by attachment to an imino-diacetate or tris(carboxymethyl)ethylenediamine substituted agarose.
4. Dye Ligand Chromatography
- Utilizes a variety of triazine dyes for ligands.
- The most popular color is Cibracron Blue F3G-A.
- It is used to purify interferons and lipoproteins as well as factors that cause coagulation, etc.
5. Covalent chromatography
- Specially designed to separate proteins containing thiol
- The most frequently used ligand an adiquate 2′-pyridyl group
- It is used to purify many proteins, however its application is restricted due to its price and difficult regeneration.
Types of affinity media used in Affinity Chromatography
A variety of affinity media are available to serve a range of applications. Briefly, they are (generalized) activated/functionalized that work as a functional spacer, support matrix, and eliminates handling of toxic reagents.
- Amino acid media: It is used in conjunction with a variety of proteins from serum enzymes, and peptides in addition to dsDNA and rRNA.
- Avidin biotin media: Avidin biotin media is used to purify the process of biotin/avidin, as well as their derivatives.
- 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
- The dye ligand medium is nonspecific however it mimics biological substrates as well as proteins.
- Hydrophobic interaction medium are frequently employed to attack free carboxyl groups and proteins.
- Immunoaffinity media uses antigens’ as well as antibodies that have high specificity to differentiate immobilized metal affinity chromatography is further described below. It uses interactions between proteins and metal ions (usually specifically labeled) to separate. Nucleotide/coenzyme which helps separate dehydrogenases, kinases and transaminases.
- Speciality media are made for specific classes or types of co enzyme. This kind of media can only function to isolate a particular type of protein, or coenzyme.
What is Weak affinity chromatography?
- WAC is a weak affinity technique. (WAC) is an affinity chromatography technique used for testing affinity for drug development.
- WAC is an affinity-based technique for liquid chromatography which separates chemical compounds according to their weak affinities with the immobilized object.
- The greater affinity a compound has with the target the longer it will remain within the separator and this is measured as a longer retention time.
- The measurement of affinity and rank of affinity are accomplished by processing the retention times of the compounds being studied.
- Affinity chromatography is a part of a wider set of chemoproteomics techniques for the purpose of identifying drug targets.
- The WAC technology has been demonstrated against various protein targets , including proteases chaperones, kinases, along with protein-protein interaction (PPI) specific targets. WAC has been proven to be more efficient than traditional methods for screening based on fragments.
Application of Affinity Chromatography
- It is used to isolate and purification of all biological macromolecules.
- It is used to purify nucleic acid,antibodies,enzymes.etc
- To determine which compounds in the biological world are bound to a specific substance.
- To decrease the amount of substance present in a mix.
- Utilized for Genetic Engineering for nucleic acid purification
- Utilized for the Production of Vaccines – antibody purification from blood serum
- It is used for Basic Metabolic Research such as the purification of enzymes or proteins from cells free extracts.
- Affinity chromatography also serves to get rid of particular contaminants, like that of Benzamidine. Sepharose(tm) 6 Fast Flow removes serine proteases like the Factor Xa and thrombin. Figure 2 shows the main phases of an affinity purification.
Advantages of Affinity Chromatography
- Extremely high-specificity
- The purest of levels can be achieved
- The process is highly reproducible.
- The binding sites of biological molecules could be investigated by simply looking at the binding sites of biological molecules.
- Single-step purification.
- The matrix is reusable in a short time.
- The matrix is solid that is easy to clean and dried.
- Provide purified products with high yield.
- Affinity chromatography may also be used to get rid of particular contaminants, for instance proteases.
Disadvantages of Affinity Chromatography
- Expensive ligands
- Leakage of the ligand
- Degradation of the solid support
- Relatively low productivity
- Non-specific adsorption can not be totally eliminated, it can only be minimized.
- The limited life span and the high cost for immobilized ligands.
- Proteins are denatured when the necessary pH is not maintained.