3 疏水层析.ppt

Read me! Hydrophobic interaction chromatography Hydrophobic interaction chromatography Hydrophobic interaction chromatography What is hydrophobic interaction chromatography? What is it used for? Underlying principles How to do it What is it? Hydrophobic interaction chromatography is a liquid chromatography technique that separates biomolecules according to their hydrophobicity Why should I use it? Complementary to ion exchange (IEX) and gel filtration (GF) Mild, non-denaturing High selectivity High recovery Concentrating technique Hydrophobic interaction chromatography What is hydrophobic interaction chromatography? What is it used for? Underlying principles How to do it Purification of recombinant a-mannosidase from Pichia pastoris Capture of recombinant human EGF Separation of a2-macroglobulin isomers Purification of rhGM-CSF from inclusion bodies produced in E. coli HIC pro′s and con′s Hydrophobic interaction chromatography What is hydrophobic interaction chromatography? What is it used for? Underlying principles How to do it The Hofmeister series Main stages in HIC Equilibrate the gel and the sample to binding conditions Apply the sample Wash out contaminants Elute Equilibration Sample application Washing out unbound material Elution Elution Hydrophobic interaction chromatography What is hydrophobic interaction chromatography? What is it used for? Underlying principles How to do it First trial, general conditions Binding buffer: 50 mM phosphate buffer pH 7.0 with 1-1.5 M ammonium sulphate Elution buffer: 50 mM phosphate buffer pH 7.0 Gradient: 10-15 column volumes Flow rate: according to manufacturers instructions Gel: Phenyl Sepharose 6 Fast Flow (high sub) How to do it? Selecting media type of ligand degree of substitution type of base matrix Selecting adsorption condition Selecting elution conditions Unexpected results Type of ligand, examples Different selectivity of HIC media Different selectivity of HIC media Degree of s