Affinity Chromatography

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Affinity Chromatography:

Affinity Chromatography it exploits the unique property of extremely specific biological interactions to achieve separation and purification

Affinity Chromatography (AC) :

Affinity Chromatography (AC) Definition:- Is the purification of a biomolecule with respect to the specific binding of that biomolecule due to the chemical structure. Very selective Specific binding site is used to concentrate analyte on column

-The principle of affinity chromatography is based on the property of specific and non-covalent binding of proteins to other molecules, referred to as substrates or cofactors. . :

- The principle of affinity chromatography is based on the property of specific and non-covalent binding of proteins to other molecules, referred to as substrates or cofactors. . Affinity chromatography Molecule Ligand Antigen Antibody Enzyme Substrate Receptor Ligand Nucleic Acid Binding Protein Nucleic Acid Polysaccharide, glycoprotein Lectin The technique was originally developed for the purification of enzymes, but it has since been extended to nucleotides, nucleic acids, immunoglobulins, membrane receptors and even to whole cells and cell fragments

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The technique involves the use of ligands covalently attached to an inert and porous matrix in a column. The immobilized ligands act as molecular hooks to selectively pick up the desired protein while the remaining proteins pass through the column. The desired protein captured by the ligands, can be eluted by using free ligand molecules. Alternatively, some reagents that can break protein ligand interactions can also be employed for the separation

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The optimum length of this spacer arm is six to ten carbon atoms or their equivalent.

METHODOLOGY:

METHODOLOGY Binding of the selected ligand to the matrix requires that a covalent bond be formed between the two. The ligand -matrix gel is then loaded into an elution column. Once the column has been prepared, the mixture containing your favorite isolate is poured into the elution column In order to remove these unbound impurities, a wash of extreme pH, salt concentration, or temperature is run through the gel Finally to collect your favorite isolate, which is still bound to the ligand -matrix in the gel, a stronger second wash is run through the column.  The protein is then free to run through the gel and be collected.

How do they get those eddy bitty molecules in there?:

How do they get those eddy bitty molecules in there?

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Immunoaffinity chromatography By using antibodies, antigens can be easily separated. Conversely, antibodies can be purified by passing through a column containing the antigen. Specific Antibody binds to a specific antigen attached to the stationary medium, the remaining antibodies come out in earlier fractions. 9 Biochemistry of Medics

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Stages in affinity chromatography The affinity chromatography process can be separated into 3 main stages: Equilibration Application of Sample Elution

So now what….:

The Sample is injected into the equilibrated affinity chromatography column Only the substance with affinity for the ligand are retained on the column The substance with no affinity to the ligand will elute off The substances retained in the column can be eluted off by changing the pH of salt or organic solvent concentration of the eluent So now what….

Specificity of Affinity Chromatography:

Specificity of Affinity Chromatography Specificity is based on three aspect of affinity 1) the matrix 2) the ligand 3) the attachment of the ligands to the matrix

Matrix:

Matrix The matrix simply provides a porous structure to increase the surface area to which the molecule can bind This has been what kept the Affinity Chromatography from being developed earlier and useful to the scientific community The matrix must be activated for the ligand to bind to it but still able to retain it’s own activation towards the target molecule

Matrix:

Matrix Amino, hydroxyl, carbonyl and thiol groups located with the matrix serve as ligand binding sites Matrix are made up of agarose and other polysaccharides The matrix also must be able to withstand the decontamination process of rinsing with sodium hydroxide or urea

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In practice, particles that are uniform, spherical and rigid are used. The most common ones are the cross-linked dextrans and agarose, polyacrylamide, polymethacrylate, polystyrene, cellulose and silica

Ligand:

Ligand The Ligand binds only to the desired molecule within the solution The ligand attaches to the matrix which is made up of an inert substance The ligand should only interact with the desired molecule and form a temporary bond

Ligand:

Ligand The ligand/molecule complex will remain in the column, eluting everything else off The ligand/molecule complex dissociates by changing the pH

MERITS:

MERITS High selectivity compared to other purification techniques based on molecular size, charge, isoelectric point,etc. Good purification upto several thousand folds in a single step and recoveries greater than 90% can be expected provided conditions are carefully selected. High concentrating effect. Can be used to remove unwanted materials from a mixture High-yield one-step purifications can be achieved from crude extracts.

DEMERITS:

DEMERITS One of the disadvantage is that a different stationary phase must be used for almost every separation. Leakage of ligand Expensive method as it involves ligands. Problem in scale up High Labour intensity

APPLICATIONS:

APPLICATIONS Purification, determination or removal of many biologically active substances. Affinity chromatography is useful for the purification of enzymes, vitamins, nucleic acids, drugs , hormone receptors, antibodies etc. The active sites of enzymes or antibodies, the binding properties of subunits, the specificity of enzymes towards various inhibitors, the complementarity of nucleic acids, the interactions of nucleotides with peptides, the effect of the presence of various substances on the formation of specific complexes, etc., can be studied by bio-affinity chromatography. Messenger RNA, for example, is routinely isolated by selective hybridisation on poly(U)-Sepharose 4B by exploiting its poly(A) tail Immobilised single-stranded DNA can be used to isolate complementary RNA and DNA

Covalent chromatography:

Covalent chromatography to separate thiol (–SH)- containing proteins by exploiting their interaction with an immobilised ligand containing a disulphide group. The method has been used successfully for many proteins but its use is limited by its cost and the rather difficult regeneration stage. It can, however, be applied to very impure protein preparations.

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Immobilized metal ion affinity chromatography (IMAC)

Principle :

Principle a special form of affinity chromatography an immobilised metal ion such as Cu2+, Zn2+, Hg2+ or Cd2+ or a transition metal ion such as Co2+, Ni2+ or Mn2+ is used to bind proteins selectively by reaction with imidazole groups of histidine residues, thiol groups in cysteine residues and indole groups in tryptophan residues sterically available on the surface of the proteins.

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this technique is the coordination between the electron donor groups on a protein (peptide) surface and immobilized transition metal ions. electron donor groups Spacer immobilized transition metal ions

Regeneration of column:

Regeneration of column

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Summary 28 Biochemistry of Medics

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