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22 Fractogel® EMD Tentacle Phases for Hydrophobic Interaction Chromatography High performance hydrophobic interaction chromatography is a powerful technique for the isolation of proteins on an analytical and preparative scale. The method of hydrophobic interaction chromatography (HIC) is based on the interaction between hydrophobic ligands on the chromatographic support and hydrophobic areas located on the surface of proteins. The hydrophobic feature of a given protein is caused by certain amino acid residues like isoleucine, valine, leucine, phenylalanine and others. Although most of the hydrophobic amino acid residues are located in the center of the molecule, recent structure analyses have shown hydrophobic patches on the protein’s surface. These areas are often correlated with the biological function of the protein. The hydrophobic character of a protein is promoted by high salt and increased temperatures. Advantages of HIC tentacle materials The high selectivity of Fractogel® EMD Propyl (S) Metal Chelate Affinity Chromatography on Fractogel® EMD Chelate Advantages of tentacle media for metal chelate affinity chromatography The high efficiency of the method is based on the interaction of a covalently bound chelating ligand immobilized on a chromatographic support with proteins containing a large number of histidine residues. Some recombinant proteins have His tags engineered at the end of the protein sequence to facilitate purification on IMAC resins. Iminodiacetic acid has been chosen as the metal-chelating ligand for Fractogel® EMD Chelate. The iminodiacetic acid residue is suitable as a chelating compound because a bidentate chelating moiety remains free after immobilization, onto which a metal ion can be co-ordinated. Various metal ions can be immobilized on the stationary phase (e.g. Cu 2+ ; Ni 2+ ; Co 2+ ; Zn 2+ ). Free coordination sites of the metal ions are used to bind different proteins and peptides. Compared to conventional affinity gels synthesised with spacer molecules, Fractogel® EMD and Fractogel® EMD Phenyl (S) allow efficient purification steps. Both phases, with a particle size of 20-40 µm, are mechanically stable and resistant to biological and chemical degradation. The functional groups are on linear polymer chains. The ligands differ with respect to their hydrophobicity. Ligand density of the functional groups bound to the matrix is optimised by the tentacle technology so that excessive hydrophobicity is avoided. Strong hydrophobic resins inhibit recovery and resolution. Application areas Both hydrophobic phases are suitable for a wide range of applications. The main application areas for HIC is the purification of cytosolic proteins, antibodies, and recombinant proteins. Membrane proteins can also be isolated on Fractogel® EMD Propyl (due to its weak hydrophobicity this resin is more suitable). For the elution of strong hydrophobic proteins, a detergent should be used. In addition HIC is suitable for the removal or exchange of non-ionic detergents. Chelate allows the binding of larger amounts of proteins. Because the immobilized metal ion can be arranged in the best position relative to the binding sites on the protein’s surface, proteins can be bound more tightly to Fractogel® EMD Chelate than comparable conventional gels. Application areas The existence of surface-accessible histidine residues is important for the binding of a native protein. As recombinant proteins become more important, expression systems using a polyhistidine tag for the protein enable rapid isolation of those proteins by metal chelating affinity chromatography. With peptides, the alpha amino group also plays a role, so that peptides can be retained even if no histidine residues are present. In the case of peptides, other metal binding amino acids like cysteine and tryptophan contribute to the binding.
Thiophilic Adsorption Chromatography on Fractogel® EMD TA Advantages of tentacle-mediated thiophilic adsorption The binding mechanism of thiophilic adsorption chromatography is based on the specific interaction of a covalently bound sulphur-containing ligand immobilized on a chromatographic support with proteins or peptides. An electron donor-acceptor mechanism between the affinity ligand and tryptophan residues on the surface of the target molecule is responsible for the selective binding. Fractogel® EMD TA is designed specifically for the efficient isolation of antibodies. The synthesis is performed according to the tentacle technology where the group-specific ligands are present in high density. Thus, the thiophilic tentacle material has a high protein-binding capacity and is suitable for antibody purification on an analytical as well as a preparative scale. Due to the steric accessibility of the ligand, the resin provides minimized non-specific interactions with proteins combined with high protein binding capacities. The chemistry of the 3-S type ligand that is used for Fractogel® EMD TA allows significantly better thiophilic binding compared to the 2-S types which have more hydrophobic interaction. Application areas Thiophilic adsorption chromatography is specifically designed for the purification of immunoglobulins (monoclonal and polyclonal antibodies) including single-chain antibodies and chicken antibodies. Albumins are not adsorbed on thiophilic media, which often simplifies the effective separation of antibodies. The binding of the protein takes place mainly via accessible tryptophan and/or phenylalanine residues. Corresponding motifs can be observed within conserved regions of various antibodies. Antibodies of the IgM subclass can also bind to a Fractogel® EMD TA column. The binding of antibodies from different species to thiophilic adsorption supports offers advantages over protein A. Elution conditions at physiological pH values enable high recoveries of biologically active antibodies. Fractogel® EMD tentacle gels for HIC / Ordering information Designation Cat.-No. Particle size Content Chromatography type: weak HIC Fractogel ® EMD Propyl (S) Chromatography type: strong HIC Fractogel ® EMD Phenyl (S) 1.10085.0100 100 ml 1.10085.0500 20 – 40 µm 500 ml 1.16197.0100 1.16197.0500 20 – 40 µm 100 ml 500 ml Bulk quantities on request. Fractogel® EMD tentacle affinity gels / Ordering information Designation Cat.-No. Particle size Content Chromatography type: metal affinity chromatography Fractogel ® 1.10338.0250 250 ml EMD Chelate (M) 1.10338.0500 40 – 90 µm 500 ml 1.10338.5000 5000 ml Chromatography type: thiophilic adsorption Fractogel ® EMD TA (S) Chromatography type: affinity chromatography Fractogel® EMD Amino (M) 1.16473.0025 25 ml 1.16473.0250 20 – 40 µm 250 ml 1.16473.1000 1000 ml 1.14893.0500 1.14893.5000 40 – 90 µm 500 ml 5000 ml Bulk quantities on request. Amino Acids Buffers Benzonase® Biochromatography Process and Production Chromatography Preparative Columns Biochemicals Enzymes 23
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