B6PROTEINPURIFICATION课稿.ppt

B6 PROTEIN PURIFICATION Principles of proteinpuri?cation(蛋白质纯化原则) The aim of protein puri?cation is to isolate one particular protein from all the others in the starting material. A combination of fractionation techniques is used that exploits the solubility, size, charge, hydrophobicity or/and speci?c binding af?nity of the protein of interest. Selection of a protein source蛋白质原料的选择 Because proteins have different distributions in biological materials, it is important to make the right choice of starting material from which to purify the protein. This will usually be a source that is relatively rich in the protein of interest and which is readily available. The use of recombinant DNA techniques means that large amounts of normally scarce proteins can be obtained by expression in bacterial or eukaryotic cells. Homogenization and solubilization匀浆和溶解 The protein has to be obtained in solution prior to its puri?cation. Thus tissues and cells must be disrupted by homogenization or osmotic lysis and then subjected to differential centrifugation to isolate the subcellular fraction in which the protein is located. For membrane-bound proteins, the membrane structure has to be solubilized with a detergent to liberate the protein. Stabilization of proteins蛋白质的稳定作用 Certain precautions have to be taken in order to prevent proteins being denatured or inactivated during puri?cation by physical or biological factors. These include buffering the pH of the solutions, undertaking the procedures at a low temperature and including protease inhibitors to prevent unwanted proteolysis. Assay of proteins蛋白质的测定 In order to monitor the progress of the puri?cation of a protein, it is necessary to have an assay for it. Depending on the protein, the assay may involve measuring the enzyme activity or ligand-binding properties or may quantify the protein present using antibodies directed against it. Ammonium sulfate precipitation硫酸铵沉淀法 The solubility of proteins decrea