Cell paste with over expressed Target Protein
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Suspend in appropriate buffer
Disruption (sonication or French-Press)
Spin down debris
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50 mM buffer pH 8.0
Centrifugation 30000-70000 g , 10-15 min.
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Crude extract
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Ion exchange chromatography
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anion exchange for acidic protein (column with DEAE-Sepharose or Q-Sepharose)
cation exchange for basic proteins (column CM-Sepharose or SP- Sepharose) (optional)
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SDS-PAGE analysis of elution profile, pooling of fractions containing Target Protein
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Ammonium sulphate precipitation analytical trial
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Protein precipitates with lower than 1.8 M AmSO4
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Protein precipitates with higher than 1.8 M AmSO4
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Precipitate protein with AmSO4
(using lowest possible concentration of AmSO4)
Collect precipitate by centrifugation and dissolve in small volume of buffer
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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.
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If OK
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If not OK
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Run full scale hydrophobic chromatography; Collect Target Protein; concentrate using concentrator or precipitate with AmSO4
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AmSO4 cut
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Gel filtration
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Run gel filtration in appropriate buffer
Analyse elution profile by SDS-PAGE
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If purity is higher than 85%
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If purity is less than 85%
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Concentrate protein using concentrator and exchange buffer on the concentrator or by dialysis to low salt
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Try Ion exchange chromatography on column with different matrix at a different pH or HPLC column
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Crystallization trials
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