2 Affinity Chromatography: Separation of proteins by reversible interaction of proteins and specific ligand in matrix of columnAdvantages:SelectivityResolutionSuitable for concentrating a protein
3 Some common terms : Matrix Spacer Arm Ligand Binding Elution Wash Ligand coupling
4 Matrix Open and porous structure Inert or non-specific interaction OH group on sugar residues , good anchor for ligand attachmentAble to pass liquids through itself rapidlyStable enough in various conditionsSepharose is an ideal matrix!
10 Different types of Affinity Chromatography Type of columnTargetEnzymeSubstrate analogue, cofactor, inhibitorImmunoaffinityAntigen, virus,cellLectinPolysaccharides, glyco-proteins, cell surface receptor, cellNucleic acidComplementary base sequence, histones, nucleic acid polymerase, nucleic acid binding proteinHormone, vitaminReceptor, carrier proteinGlutathioneGlutathione-S-transferase, GST fusion proteinImmobilized metal ion affinity chromatography (IMAC)Poly (his)fusion proteins, native proteins with histidine, cystein or tryptophan residues on their surfaces
11 Immuno-affinity chromatography (IAC) A sub category of affinity chromatographyA biologically related binding agent is used for the selective purification or analysis of a target compoundstationary phase consists of an antibody or antibody-related reagentThe selective and strong binding of antibodies for their given targets has made them of great interest as immobilized ligands in affinity chromatography
12 Moser A.C., et. Al., Immunoaffinity chromatography: an introduction to applications and recent developments, bioanalysis ,2(4):
13 The loading buffer has the ability to promote fast and efficient binding of the target analyte to immobilized antibodies which typically occurs under physiological conditions pH=7The elution carry out by temporarily lowering the effective strength of antibody binding to the target.Changing the mobile phase pH is the most popular method for eluting retained compounds from IAC columns.This approach is usually conducted by applying an acidic buffer (pH 1–3) to the column
14 An example of competitive elution: http://youtu.be/Hb791WsC78s