Affinity chromatography (AC) is a powerful method that relies on the principle of molecular recognition, exploiting the ability of various substances and biological compounds to form specific pairs, either reversibly or irreversibly. Attaching one reactive component to a solid-phase substrate allows the capture of a second interacting substance under mild conditions, even from the complex biological materials. When optimal elution conditions are adjusted, specifically captured molecules are quantitatively released from the affinity matrix, achieving them in a high purity. A well-designed affinity matrix combined with optimized separation conditions facilitates the acquisition of bioactive molecules in their native form. Given the wide range of substances suitable for affinity isolation, this article introduces the readers to the basics of protein AC, with the intention to provide a practical guide on how to prepare the affinity matrix and apply it for the effective isolation and purification of protein molecules.

• Klíčová slova
• Affinity matrix, Immobilization, Molecular recognition, Protein affinity chromatography, Protein purification, Specific ligand,
• MeSH
• chromatografie afinitní * metody   přístrojové vybavení   MeSH
• lidé MeSH
• proteiny * izolace a purifikace   chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• proteiny * MeSH

Over the past decades, the proteomics field has undergone rapid growth. Progress in mass spectrometry and bioinformatics, together with separation methods, has brought many innovative approaches to the study of the molecular biology of the cell. The potential of affinity chromatography was recognized immediately after its first application in proteomics, and since that time, it has become one of the cornerstones of many proteomic protocols. Indeed, this chromatographic technique exploiting the specific binding between two molecules has been employed for numerous purposes, from selective removal of interfering (over)abundant proteins or enrichment of scarce biomarkers in complex biological samples to mapping the post-translational modifications and protein interactions with other proteins, nucleic acids or biologically active small molecules. This review presents a comprehensive survey of this versatile analytical tool in current proteomics. To navigate the reader, the haphazard space of affinity separations is classified according to the experiment's aims and the separated molecule's nature. Different types of available ligands and experimental strategies are discussed in further detail for each of the mentioned procedures.

• Klíčová slova
• Affinity chromatography, Affinity depletion, Affinity enrichment, Mass spectrometry, Peptide, Protein, Proteomics,
• MeSH
• chromatografie afinitní * metody   MeSH
• lidé MeSH
• proteiny izolace a purifikace   analýza   chemie   MeSH
• proteomika * metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• proteiny MeSH

The authors studied the separation of glucoamylase and beta-galactosidases from the interstinal mucosa of young rats by affinity chromatography. They tested the following chromatographic materials: p-aminophenyl-beta-D-thioglucoside bound to Sepharose 4B via hexamethylenediamine, gluconate and galactonate bound in different ways to Sepharose 4B and phlorizin bound by an azo-coupling reaction to a spacer attached to Sepharose 4B. The conditions of the adsorption of glycosidases to these materials and their subsequent elution were studied. Using chromatography on Sepharose 4B with a beta-thioglucoside affinant, we succeeded in purifying lactase preparation so that, in electrophoresis on polyacrylamide gel, it formed a single zone identical with 1-naphthyl-beta-glucosidase activity.

• MeSH
• chromatografie afinitní metody   MeSH
• glukosidy MeSH
• glykosidhydrolasy izolace a purifikace   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glukosidy MeSH
• glykosidhydrolasy MeSH

Affinity purification, combined with mass spectrometry (AP-MS) is considered a pivotal technique in protein-protein interaction studies enabling systematic detection at near physiological conditions. The addition of a quantitative proteomic method, like SILAC metabolic labeling, allows the elimination of non-specifically bound contaminants which greatly increases the confidence of the identified interaction partners. Compared to eukaryotic cells, the SILAC labeling of bacteria has specificities that must be considered. The protocol presented here describes the labeling of bacterial cultures with stable isotope-labeled amino acids, purification of an affinity-tagged protein, and sample preparation for MS measurement. Finally, we discuss the analysis and interpretation of MS data to identify and select the specific partners interacting with the protein of interest. As an example, this workflow is applied to the discovery of potential interaction partners of glyceraldehyde-3-phosphate dehydrogenase in the gram-negative bacterium Francisella tularensis.

• Klíčová slova
• Affinity purification, Bacteria, LC-MS/MS, Protein-protein interactions, SILAC,
• MeSH
• Bacteria metabolismus   MeSH
• chromatografie afinitní MeSH
• hmotnostní spektrometrie metody   MeSH
• izotopové značení metody   MeSH
• proteiny * chemie   MeSH
• proteomika * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny * MeSH

Immobilization of affinity ligands, proteins, enzymes and other functional groups by azo coupling is based on the high reactivity of the support-carrying diazonium groups towards both low- and high-molecular weight compounds containing certain groupings such as phenols, imidazole and some other heterocycles, thiols and amines. The precursor of diazonium group is a diazotizable amine on the matrix. Remarkable progress in its preparation was achieved by application of (4-amino-phenyl)-(2-sulphatoxyethyl) sulphone-type reagents for functionalization of the matrix. A similar type of amine precursor is prepared by the reaction of monosubstituted sulphanilamide with epoxide-containing matrix. The presence of a SO2 group in the para position to diazonium group of these supports (after diazotization) enhances reactivity in azo coupling. 'Reversed' azo coupling is the reaction of a matrix containing functional groups capable of reacting with diazonium groups of a ligand. Preparation of a suitable matrix and examples of diazotizable ligands are given.

• MeSH
• chromatografie afinitní * MeSH
• diazoniové sloučeniny chemie   MeSH
• ligandy MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• diazoniové sloučeniny MeSH
• ligandy MeSH

Three stationary phases have been prepared for affinity liquid chromatography isolation and separation of porcine and human pepsin. The phases contain 3,5-diiodo-L-tyrosine (DIT) bound to the supports HEMA BIO VS, HEMA BIO E and EPOXY TOYOPEARL. These phases have been tested on a model sample of porcine pepsin A and applied to human pepsin. Fractions have been collected and the chymase activity determined in selected analyses. For affinity CE, capillaries have been prepared by modifying the wall with 3-aminopropyltriethoxysilane, followed either by direct binding of DIT, or by binding L-tyrosine that was subsequently iodated. The dissociation constant K(d) has been determined for the pepsin-DIT complex from the changes in the electrophoretic mobilities.

• MeSH
• chromatografie afinitní přístrojové vybavení   metody   MeSH
• dijodtyrosin chemie   MeSH
• elektroforéza kapilární přístrojové vybavení   metody   MeSH
• lidé MeSH
• oxid křemičitý chemie   MeSH
• pepsin A izolace a purifikace   MeSH
• prasata MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dijodtyrosin MeSH
• oxid křemičitý MeSH
• pepsin A MeSH

Stationary phase containing quinolin-8-ol immobilized on macroporous methacrylate support for the affinity chromatography of porcine pepsin A is described. Optimized chromatographic conditions for separation of porcine pepsin A on this stationary phase were found investigating the influence of pH, concentration, ionic strength and chemical composition of the used mobile phases. The stationary phase shows a good reproducibility of chromatographic analyses (relative standard deviation, +/-2%), a high recovery (ca. 93%) and a satisfactory capacity (13 mg pepsin A/1 mL stationary phase) for porcine pepsin A. The obtained findings confirm the applicability of affinity chromatography on the stationary phase with immobilized quinolin-8-ol to the isolation and determination of porcine pepsin A.

• MeSH
• chromatografie afinitní metody   MeSH
• hydroxychinoliny chemie   MeSH
• ligandy MeSH
• pepsin A izolace a purifikace   MeSH
• prasata MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hydroxychinoliny MeSH
• ligandy MeSH
• pepsin A MeSH

The preparation of affinity sorbents containing immobilized iodinated derivatives of L-tyrosine for the affinity chromatography of porcine pepsin is described. The ligand was coupled either to Sepharose 4B or bead cellulose after the divinylsulfone activation or to Sepharose 4B after the activation with 2,4,6-trichloro-1,3,5-triazine. The highest capacity for porcine pepsin was found in the case of 3,5-diiodo-L-tyrosine coupled to divinylsulfone-activated Sepharose.

• MeSH
• chromatografie afinitní metody   MeSH
• dijodtyrosin chemie   MeSH
• prasata MeSH
• triaziny chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyanuric chloride MeSH Prohlížeč
• dijodtyrosin MeSH
• triaziny MeSH

The aim of this work was to isolate plant ecdysteroid-binding proteins using affinity chromatography. Ecdysteroids as insect hormones have been investigated thoroughly but their function and the mechanism of action in plants and other organisms is still unknown although ecdysteroids occur in some plants in a relatively large amount. Therefore, 20-hydroxyecdysone was immobilized on a polymeric carrier as a ligand for affinity chromatography in order to isolate plant ecdysteroid-binding proteins from the cytosolic extract of New Zealand spinach (Tetragonia tetragonoides). Non-specifically bound proteins were eluted with a rising gradient of concentration of sodium chloride, and 3% (v/v) acetic acid was used for the elution of the specifically bound proteins. Using this method, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was isolated. The influence of ecdysteroids on RuBisCO was further studied. Our results show that ecdysteroids are able to increase the yield of RuBisCO-mediated reaction in which CO(2) is fixed into organic matter by more than 10%.

• MeSH
• chromatografie afinitní * MeSH
• cytosol enzymologie   MeSH
• ekdysteron metabolismus   MeSH
• enzymy imobilizované MeSH
• ribulosa-1,5-bisfosfát-karboxylasa izolace a purifikace   metabolismus   MeSH
• Spinacia oleracea enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ekdysteron MeSH
• enzymy imobilizované MeSH
• ribulosa-1,5-bisfosfát-karboxylasa MeSH

Natural antibodies are now widely studied for their therapeutical potential. Therefore, their isolation and subsequent characterization is desired. Here, we describe an isolation method for natural anti-tau antibodies from human plasma by utilization of affinity chromatography with epoxy-activated copolymer resin. The evalution of isolation efficiency and avidity of isolated antibodies is decribed by modified indirect ELISA assay.

• Klíčová slova
• Human plasma, Immobilization, Immunopurification, Immunoreactivity, Intravenous immunoglobulin, Natural antibodies, Tau protein,
• MeSH
• autoprotilátky imunologie   izolace a purifikace   MeSH
• chromatografie afinitní * MeSH
• imunosorpční techniky MeSH
• intravenózní imunoglobuliny MeSH
• lidé MeSH
• proteiny tau imunologie   MeSH
• protilátky imunologie   izolace a purifikace   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• autoprotilátky MeSH
• intravenózní imunoglobuliny MeSH
• proteiny tau MeSH
• protilátky MeSH