GE-双向电泳的样品制备.ppt

Title or job number 双向电泳的样品制备 样品的准备步骤 Cell disruption Protein precipitation Solubilization Protection against protease activities Removal of nucleic acids lipids salts, buffers, ionic small molecules insoluble material 在样品制备前需要考虑的问题 Is the sample from cells or solid tissue? Is pre-fractionation desired? What kind of interfering substances are present? Quality of separation vs. total protein representation 细胞破碎的方法 Freeze-thaw or osmotic lysis Detergent lysis Sonication Enzymatic lysis French pressure cell Grinding (mortar and pestle) Mechanical homogenization 现在常用在样品预分离的方法 By solubility sequential extraction with increasingly strong solubilizing agents (???) By chromatography e.g. hydrophobic interaction (???) By centrifugal separation of subcellular components e.g. mitochondria, nuclei, cytosol By affinity Immunoprecipitation of complexes or selective removal of abundant proteins 干扰物质 Proteases Nucleic acids Polysaccharides Plant phenols Lipids Salt ions Insoluble material 如何使蛋白酶失活 Problem: Cell lysis → endogenous proteases set free → proteolytic attack → artifacts (Mr, pI) 常用的蛋白酶抑制剂 PMSF inhibits serin- and thiol-proteases - is not stable in aqueous solutions - is inactivated by DTT, ?-mercaptoethanol etc. - is toxic (alternative: Pefablock?) AEBSF (Pefabloc?) alters pI of some proteins EDTA inhibits metallo-proteases Pepstatin inhibits acidic proteases high pH inhibits/slows down actions of proteases Limits: Protease inhibitors do not inactivated all proteases! Proteins may become modified by protease inhibitors 核酸和多糖 cause horizontal and vertical streaks in 2-D patterns increase sample viscosity clog pores of PAGE may cause background smear in silver stained 2-D patterns Removal of nucleic acids: treatment with a protease-free DNAse/RNAse mixture TCA/acetone precipitation of proteins and re-solubilization adding a basic polyamine (e.g. spermine) and ultracentrifugation Removal of polysaccharides: TCA/acetone preci