Biochem purification methods

Salting out

separation by protein solubility in high ammonium acetate

Chromatography

Separation based on ionic charge, polarity, size, binding ability

Gel electrophoresis

separates according to size, charge, isoelectric point

Ultracentrifugation

Based on overall size and shape without denaturing the protein

Fractionation

Ion exchange chromatography

Positively charged proteins bind to cation (+)

Negatively charged proteins bind to anions (-)

Proteins released by high salt concentration

Gel filtration

Larger proteins leave first, they have no access to matrix of gel beads.

Dialysis

Sample placed in a bag, bag immersed in solution, diffusible solutes in bag go across membrane while protein stays in the bag.

Salt ions removed before ion exchange step

Affinity chromatography

Small molecule targets are immobilized through covalent attachment to solid matrix in column

• Protein passed through column and binds to target ligands, other proteins pass without binding

• Protein eluted by addition of high conc. of unbound ligand

SDS PAGE

• Denatures proteins with SDS detergent and binds to them to make them uniformly (-).

• When electric current is applied, they all move to the positive side

• Smaller proteins move farther than bigger proteins

• when the sample has been separated for a specific amount of time, they can be stained

• Reducing agents: DTT or beta Me