Timing for soluble protein purification procedure
Main steps:
- Protein purification- Introduction
- Step-by-Step
- Common sense
- Common sense strategy
- Purification protocol
- Purification of soluble protein
- Purification of DNA-binding protein
- Timing of purification
- Protocols
- Charts & Tables
- Appendix
- Crystallisation
Special:
- Programs for crystallography- X-ray detectors
Basic tutorials:
- Chemistry- Protein
- Peptide
- Amino Acids
Xtal community:
- CCP4BBProtocols and tips in protein purification or How to purify protein in one day.
Step-by-step protein purification timing
Preparation of the crude extract
|
1 hour |
|
Ion-exchange chromatography on DEAE-Sepharose Fast Flow
|
1-2 hours |
|
SDS-PAGE (optional)
|
1.5 hours |
|
Ammonium sulphate cut or preparation of sample for hydrophobic chromatography in 1.5M - 1.8M AmSO4
|
0.5-1 hour |
|
Hydrophobic chromatography on Butyl, Phenyl or Ether-Toyopearl 650S
|
1-2 hours |
|
Concentration on VivaSpin concentrators or Ammonium sulphate precipitation for storage or to prepare a sample for gel filtration
|
0.5-1 hour |
|
Gel filtration
|
2 hours |
|
SDS-PAGE analysis
|
1.5 hours |
| Concentration and preparation for storage or use | 1 hour |
The whole procedure takes from 5 hours (for two steps procedure) to 13 hours (for a 3 step procedure with SDS-PAGE after first step)