Protein purification and separation techniques

Gel filtration chromotography

• Elute based on size

• larger proteins elute first

• smaller proteins elute last because they are slowed down by entering the beads

Ion-exchange chromatography

• purification and seperation based on charge

• Anion exchange - positive proteins elute first, bcuz anions are binded

• cation exchange - negative proteins elute first, bcuz cations are binded

• pI of protein must be known 

Affinity chromatography

• affinity tag

• target protein interacts with ligand that is bound to the resin

• non target proteins elute first, leaving only target protein in the column

• target proteins are then eluted using the competing ligand

High Performance Liquid Chromatography (HPLC)

• separation of small molecules

• version of gel filtration

Gel electrophoresis

• NOT a purification, but a separation technique

• separation based on size/charge ratio

• larger proteins move slower

SDS-PAGE

• NOT a purification technique, but seperation technique

• used to separate proteins based on mass

• large proteins move slower

• protein is denatured (cannot be recovered)

• useful for size and purity determination of sample

isoelectric focusing

• separates proteins based on pI

• Cations will migrate towards the cathode and anions will migrate towards the anode

• will migrate until they have no net charge (pH = pI)

2D gel electrophoresis

• used to separate proteins based on both pI and molecular mass

• protein spots can be excised using mass spec or Edman degradation

• Isoelectric focusing combined with SDS-PAGE

• can separate thousands of proteins into discrete spots

2D differential In-Gel electrophoresis (DIGE)

• uses fluorescent dyes to distinguish two proteins run on the same SD-PAGE gel

1 subunit, 2 subunit, 3 subunit

monomer, dimer, trimer