色谱双语课件.ppt

酶底物，激素-受体 * ：蛋白质表面一般有疏水与亲水集团，疏水层析是利用蛋白质表面某一部分具有疏水性，与带有疏水性的载体在高盐浓度时结合。在洗脱时，将盐浓度逐渐降低，因其疏水性不同而逐个地先后被洗脱而纯化，可用于分离其它方法不易纯化的蛋白质。 * Ion Exchange chromatography (IEX) Third step --- elution Ion Exchange chromatography (IEX) Third step --- elution Ion Exchange chromatography (IEX) Final step --- washing Ion Exchange chromatography (IEX) Re- -equilibration Use some very low ionic buffer (usually use Deionized water ) to regenerate the media Gel filtration/Size exclusion Gel filtration separates molecules according to differences in size as they pass through a gel filtration medium packed in a column. Unlike ion exchange or affinity chromatography,molecules do not bind to the chromatography medium so buffer composition does not directly affect resolution Packed Medium Affinity Chromatography Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatography matrix. The technique offers high selectivity, hence high resolution, and usually high capacity for the protein(s) of interest. Affinity chromatography is unique in purification technology since it is the only technique that enables the purification of a biomolecule on the basis of its biological function or individual chemical structure. Purification that would otherwise be time-consuming, difficult or even impossible using other techniques can often be easily achieved with affinity chromatography. Affinity Chromatography Some typical biological interactions, frequently used in affinity chromatography: Enzyme ? substrate analogue, inhibitor, cofactor. Antibody ? antigen, virus, cell. Lectin ? polysaccharide, glycoprotein, cell surface receptor, cell. Nucleic acid ? complementary base sequence, histones, nucleic acid polymerase, nucleic acid binding protein. Hormone, vitamin ? receptor, carrier protein. Glutathione ? glutathione-S-transferase or GST fusion proteins. Metal ions ? Pol