Preparation of the chromatographic columns
| - Protein crystallography
Main steps:- Protein purification
- Equipment & Reagents
- Stock solutions
- Chromatographic columns
- Crude extract
- pre Chromatographic step
- Chromatographic step
- Common sense
- Charts & Tables
Special:- Programs for crystallography
- X-ray detectors
Basic tutorials:- Chemistry
- 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 with
DEAE-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.