Lecture11_proteintech课稿.ppt

The Isoelectric Points of Some Proteins Two-dimensional (2D) Electrophoresis (双向电泳) Separates proteins of identical molecular weight that differ in pI, or proteins with similar pI values but different molecular weights. 2D Electrophoresis Result Lecture 11 Protein Separation and Purification Centrifugation (离心) Chromatography (层析) Electrophoresis (电泳) Others Salting Out (盐析) Salting out： most proteins are less soluble at high salt concentrations. The salt concentration at which a protein precipitates differs from one protein to another. 0.8 M ammonium sulfate precipitates fibrinogen (纤维蛋白原), a blood-clotting protein. 2.4 M ammonium sulfate is needed to precipitate serum albumin (清蛋白；白蛋白). Salting out is also useful for concentrating dilute solutions of proteins, including active fractions obtained from other purification steps. Dialysis can be used to remove the salt if necessary. Dialysis (透析) Separate small and large molecules. Semipermeable (半通透性) membrane. Protein molecules (red) are retained within the dialysis bag, whereas small molecules (blue) diffuse. Ultrafiltration (超过滤) An improvement on the dialysis principle. Microscopic pores in the filters. Centrifugal ultrafiltration: Molecular weight cut off (MWCO; 截流分子量) The molecular weight of protein ≥5x MWCO Proteins of high molecular weight are retained, while water and low molecular weight proteins pass through the membrane. This technique is useful for concentrating dilute solutions of macromolecules. The concentrated protein can then be diluted into the solution of choice. Basic Principles of Protein Purification Ammonium sulfate fractionation Cell Organelle Homogenization Macromolecule Nucleic acid Carbohydrate (Lipid) Size Charge Polarity Affinity Small molecule Cell Debris Protein Amino acid, Sugar, Nucleotides, etc Gel filtration, SDS, Ultrafiltration Ion exchange, Chromatofocusing, Disc, Isoelectric focusing Reverse phase chromatography, HIC, Salting-out Affinity chromatography, H