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凝胶层析(生化实验)课件
Chromatography Size Exclusion Separation of proteins based on their size: The beads are composed of dextran polymers (sephadex), agarose (Sepharose), polyacrylamide (Sephacryl or BioGel P). Each bead contains pores of approximate macromolecule sizes. Larger molecules will travel through less pores, thus migrate faster. Smaller molecules will travel through more pores, thus migrate slower. The molecules passively distribute between the volume outside the porous beads (V0) and the volume inside the beads (Vi) dependent on their ability to enter the pores. If the total volume of the column is Vt, Vt = Vo + Vi Affinity Chromatography Affinity chromatography makes use of the affinity between a ligand and the protein of interests. Ligands are usually immobilized through covalent bonds on insoluble matrix, such as cellulose or polyacrylamide. The protein of interests become bound to the matrix while other proteins flow through the column. After washing the protein bound to the matrix can be eluted by adding completing groups such as the free ligand, or reagents that disrupt the interactions. Examples of ligand-protein interactions include those between antibodies and antigens, those between Ni+ and poly-histidine tag. Ion Exchange Chromatography Ion exchange chromatography makes use of electrostatic properties of the protein of interests. Charged polymers are usually immobilized through covalent bonds on insoluble matrix, such as cellulose or polyacrylamide. The protein of opposite charge become bound to the matrix while other proteins flow through the column. After washing, the protein bound to the matrix can be eluted by adding salts. Hydrophobic Interaction Hydrophobic interaction chromatography makes use of the hydrophobic patches on protein and their interactions with hydrophobic resins. For instance, phenyl sepharose is a strong hydrophobic resin that is made by covalently attach phenyl group to agarose supporting matrix. Proteins bind to phenyl group by
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