Protein purification

Activity

Total number of enzyme activity units in a solution

Specific activity (units/mg)

activity (units) / total protein (mg)

yield

total activity of your target

purity

specific activity of your target

Important considerations in protein purification

How much protein is needed? Protein source? Properties used to purify the protein?

What properties can be used to purify proteins?

Charge, size, Surface hydrophobicity, Binding affinity for a specific ligand, solubility

Protein purification when using binding affinity for a specific ligand

Tag protein of interest with a molecule through genetic engineering, express it in your system (eg bacterial cell), put the protein in a column containing immobilised ligands. This results in the protein of interest sticking to the ligand, allowing all the proteins you don't want to wash through. You can then elute the purer sample containing the protein of interest.

Liquid chromatography

Solid, porous matrix inside glass column. A buffer solution (mobile phase) flows through the matrix and elutes from the bottom of the column and is constantly replaced to keep the column wet. Sample is added to top and allowed to descend into matrix. Proteins migrate through column at different rates depending on interactions with matrix material. Individual types of proteins separate from each other.

Ion exchange chromatography

Proteins move at a rate depending on their net charge. Charged resin repels proteins with the same charge so they pass through the column faster. Proteins with an opposite charge to the resin are attracted to the resin and stay in the column longer.

Size exclusion chromatography

Proteins move through the column at a rate dependant on their size. Large proteins pass freely through the column, eluting quickly. Smaller molecules can enter the porous polymer beads - taking a longer route through the column -so elute slower.