Brief scheme of purification of soluble protein

Protein crystallography Programs for crystallography X-ray detectors
Basic tutorials:
Protein Peptide

Navigation: Go back to the Protein purification
Navigation: Go back to the Chapter 3. Common sense
Navigation: Go to: previous Development of purification protocol
Navigation: Go to: next Purification of DNA-binding proteins

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