Methods of protein purification
–Salt Precipitation –Ion-exchange –Gel filtration –Hydrophobic chromatography –Affinity chromatography
Factors affecting success in protein purification
1.Temperatures -Generally done on ice or at 4C 2. Protein Concentration and or salt concentration 3. Time (minimize)
1. Ammonium Sulfate Precipitation
Protein In water
Salting-out by (NH4)2SO4Protein in Ammonium Sulfate
Ammonium Sulfate Precipitation
Protein 1 Protein 2 Protein 3 Step 2 Step 3
- Separation ofprotein molecules based on solubility (step gradient) - Separation achieved by varying salt concentration, clarified by centrifugation
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Dialysis - removes unwanted salt from sample
2. Ion Exchange Chromatography -
Separation of protein molecules on the basis of their charge
Cation exchange chromatography: Na+ Na+
Na+ Na+ Anion exchange chromatography: Cl-
+Ion Exchange Chromatography
Separation achieved by increasing salt concentration
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Anion exchange anionscompete Cation exchange cations compete
3. Gel Filtration Chromatography
- Separate molecules on the basis of their size - Separation based on the time different molecules are entrapped by thematrix pores - Retention time inversely proportional to size
Gel Filtration Chromatography
Porous structure of agarose matrix surface4. Hydrophobic Chromatography
1. Hydrophobicity of amino acid sidechains: Trp > Ile, Phe > Tyr > Leu > Val > Met 2. Most hydrophobic side-chains are buried in interior of...
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