Methods Of Protein Purification

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

Hydrophobic interaction

Ammonium Sulfate Precipitation
Protein 1 Protein 2 Protein 3 Step 2 Step 3

solubility

Step 1

Salt Concentration

- Separation ofprotein molecules based on solubility (step gradient) - Separation achieved by varying salt concentration, clarified by centrifugation

QuickTime™ and a TIFF (Uncompressed) decompressor are neededto see this picture.

Dialysis - removes unwanted salt from sample

Column Chromatography-video

2. Ion Exchange Chromatography -

Anion Exchange

Cation Exchange

Ion ExchangeChromatography
Separation of protein molecules on the basis of their charge
Cation exchange chromatography: Na+ Na+

Na+ Na+ Anion exchange chromatography: Cl-

+

+

+ +

Cl-

Cl-

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
Cellulose

Dextran

agarose

Crosslinked polyacrylamide

Porous structure of agarose matrix surface4. 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...