Preparation of the chromatographic columns |
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- Protein crystallographyMain steps:- Protein purification- Introduction - Step-by-Step - Equipment & Reagents - Stock solutions - Chromatographic columns - Crude extract - pre Chromatographic step - Chromatographic step - Common sense - Protocols - Charts & Tables - Appendix - Crystallisation Special:- Programs for crystallography- X-ray detectors Basic tutorials:- Chemistry- Protein - Peptide - Amino Acids Xtal community:- CCP4BB |
Protocols and tips in protein purification or How to purify protein in one day. Preparation of the chromatographic columnsFor the basic purification protocol you need a set of 3 columns packed withDEAE-Sepharose Fast Flow (weak anion exchanger) Phenyl-Toyopearl 630S (hydrophobic matrix) Superdex-200 (gel filtration) The additional set includes:Q-Sepharose Fast Flow (strong anion exchanger)CM-Sepharose fast flow (weak cation exchanger) DEAE-Toyopearl 650S ( weak anion exchanger) SP-Toyopearl 650S (strong cation exchanger) Butyl-Toyopearl 650S (strong hydrophobic matrix) Ethyl-Toyopearl 650S (weak hydrophobic matrix) Heparin-Sepharose Fast Flow (pseudo-affinity) You may need other types of matrices, such as Hydroxylapatite, Dye matrix, etc. You can buy ready made columns from Amersham or Bio-Rad. For our scale we need columns 10-30 ml in size. I prefer to make them myself as this is cheaper and makes them repackable. I like Amersham-Pharmacia empty columns C-type and XK-type with adapters. The sizes are 1x20 cm and 1.6x20 cm. To prepare any column (except the gel filtration one):
A 1.6x60 cm Hi-Load Superdex 200 column (Amersham-Pharmacia) is the best option for the majority of proteins. |