色谱纯化概述.ppt

* Proteins that do not interact with the media elute in the flowthrough. After sample application all of the unbound material is washed out. If the washing step is omitted or not performed long enough (A 280 is down to zero) some of the unbound proteins will contaminate the eluate * Bound proteins are eluted by decreasing the salt concentration. Gradient elution can be used to separate the target protein from other bound proteins, normally a gradient of 10 to 15 column volumes is sufficient. * Different proteins elute at different places in the gradient. The target protein should optimally elute in the middle of the gradient. * Often you need to optimize your purification. start with media screening using the buffer conditions just described * There exist many different ligands for HIC 3 examples are shown here These ligands are included in small HIC media screening kits * From data file HiPrep HIC, figure 3. Sample volume 2 ml of 60 mg/ml in binding buffer, mixed 1:1:3:1 (of protein 1,2,3,4). Columns: HiPrep 16/10 Phenyl (high sub), HiPrep 16/10 Butyl and HiPrep 16/10 Octyl Sample: Cytochrome C (1), lysozyme (2), ribonuclease A (3) and a-chymotrypsinogen (4) Buffer A: 100 mM sodium phosphate, 1.5 M ammonium sulphate, pH 7.0 Buffer B: 100 mM sodium phosphate, pH 7.0 Flow: 2 ml/min, 60 cm/h System: ?KTAexplorer Gradient 0-100% elution buffer in 10 CV (200 ml). The different elution profiles reflect the broad selectivity of the columns and may seve as guidelines for choosing the most suitable medium for a wide range of applications. Point out that screening of these ligands can be performed using the HiTrap HIC Test Kit * Purification of a labile, oxygen-sensitive enzyme RESOURCE HIC columns were selected in order to scout for the most appropriate HIC ligand. Columns: RESOURCE ISO, RESOURCE ETH, RESOURCE PHE Sample vol: 10 ml Sample prep: DAOCS is a key enzyme involved in cephalosporin / cephamycin biosynthesis. Cloned and expressed as a soluble protein in