Chapter 5

Protein Purification and Characterization

Isolation of Proteins from cells

• Cell fractionation: proteins released from cells using physical techniques

  • blender processing or tissue sonicator

• Differential centrifugation: smaller and smaller cell components form pellet

Salting out

• soluble proteins in cytosol separated via ammonium sulfate

  • salt alters structure of water, making protein less soluble

    • hydrophobic interactions among proteins increases

    • aliquots taken as function of salt concentration

      • one fraction has protein of interest

Protein purification

• Column chromatography: different compounds distribute between phases

  • stationary: samples interact with this phase

  • mobile: flows over stationary, carries sample

    • Ion-exchange: protein separation based on charge

      • cation-exchange: negatively charged resin, exchange of cation

      • anion-exchange: positively charge resin, exchange of anion

    • size-exclusion: proteins separated by size

      • stationary phase of cross-linked gel particles

      • larger molecules elute before smaller ones

    • affinity: specific binding properties

      • stationary phase has polymer that can be covalently linked to ligand that specifically binds to protein

      • after unwanted proteins are eluted, more ligand/salt is added

        • salt weakens binding of protein to ligand

          • extra free ligand competes with column ligand

Electrophoresis

• charged particles migrate toward opposite charge

• proteins have different mobility based on charge, size, and shape

• agarose for nucleic acids, polyacrylamide for proteins

  • polyacrylamide is more resistant to larger molecules

• protein is treated with detergent (SDS) giving uniform charge

  • smaller proteins move faster