Brief scheme of purification of soluble protein

- Protein crystallography

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:

- CCP4BB

Protocols and tips in protein purification or How to purify protein in one day.

Cell paste with over expressed Target Protein

Suspend in appropriate buffer
Disruption (sonication or French-Press)
Spin down debris

50 mM buffer pH 8.0

Centrifugation 30000-70000 g , 10-15 min.

Crude extract

Ion exchange chromatography

anion exchange for acidic protein (column with DEAE-Sepharose or Q-Sepharose)
cation exchange for basic proteins (column CM-Sepharose or SP- Sepharose) (optional)

SDS-PAGE analysis of elution profile, pooling of fractions containing Target Protein

Ammonium sulphate precipitation analytical trial

Protein precipitates with lower
than 1.8 M AmSO₄

Protein precipitates with higher
than 1.8 M AmSO₄

Precipitate protein with AmSO₄

(using lowest possible concentration of AmSO₄) Collect precipitate by centrifugation and dissolve in small volume of buffer

Hydrophobic chromatography

Take about 25% of the protein sample, add 1.8 M AmSO₄ and run small scale hydrophobic chromatography. Analyse elution profile by SDS-PAGE.

If OK	If not OK
Run full scale hydrophobic chromatography; Collect Target Protein; concentrate using concentrator or precipitate with AmSO₄	AmSO₄ cut

Gel filtration

Run gel filtration in appropriate buffer
Analyse elution profile by SDS-PAGE

If purity is higher than 85%

If purity is less than 85%

Concentrate protein using concentrator and exchange buffer on the concentrator or by dialysis to low salt

Try Ion exchange chromatography on column with different matrix at a different pH or HPLC column