Hydrophobic Interaction Chromatography教程.pdfVIP

Hydrophobic Interaction Chromatography Content What is hydrophobic interaction chromatography? What is it used for? What is it used for? Interaction/Underlying principles Interaction/Underlying principles How to do it How to do it Conclusions Conclusions 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, gel filtration GF and affinity • Mild, non-denaturing • High selectivity • High recovery • Concentrating technique Content What is hydrophobic interaction What is hydrophobic interaction chromatography? chromatography? What is it used for? Interaction/Underlying principles Interaction/Underlying principles How to do it How to do it Conclusions Conclusions Purification of recombinant α-mannosidase from Pichia pastoris Technique Purification factor UF Ultrafiltration • 10 times purification AIEX Q Sepharose™ FF 63 • 82% yield HIC Phenyl Sepharose HP 622 Y.-F. Liao et al. (1996) J. Biol. Chem. 271, 28348 GF Superdex™ 200 pg 719 Capture of recombinant human EGF ™ ™ Column: BPG 300/500 packed with Phenyl Sepharose 6 Fast Flow (high sub) Sample: 80 l yeast supernatant Load: 0.36 mg EGF/ml media Buffer A: 20 mM sodium phosphate, pH 7.0 + 0.5 M