Chromatography

Question 1

Size exclusion (sometimes called gel filtration)

Answer 1

Proteins are separated by size (Molecular weight). Smaller proteins are able to pass through the channels within the resin beads and are eluted later. Larger proteins cannot fit into the channels and can only flow in between the beads and are eluted from the column faster. Normally proteins are eluted isocratically with buffer containing 0.15M NaCl to prevent non-specific ionic interactions between proteins and resin.

Question 2

Cation exchange chromatography

Answer 2

Proteins are separated by charge. The resin contains a negatively charged species eg carboxymethyl group. This will interact with positively charged amino acids on the surface of the proteins. Negatively charged proteins will not bind. Depending on how positive charge the proteins are, they will be eluted at different times in a gradient of low to high sodium chloride buffer. Buffer containing increasing concentration of sodium chloride. Sodium ions will compete with positively charged proteins for the negative charge on the resin.

Question 3

Anion exchange chromatography

Answer 3

Proteins are separated by charge. The resin contains a positively charged species eg diethylaminoethyl group. This will interact with negatively charged amino acids on the surface of the proteins. Positively charged proteins will not bind. Depending on how negative charge the proteins are, they will be eluted at different times in a gradient of low to high sodium chloride buffer. Buffer containing increasing concentration of sodium chloride. Chloride ions will compete with negatively charged proteins for the negative charge on the resin.

Question 4

Hydrophobic interaction chromatography

Answer 4

Proteins are separated by hydrophobicity. Resin contains hydrophobic groups eg phenyl groups. This will interact with proteins that have hydrophobic surfaces. Most proteins however have charged/hydrophilic groups on the surface. To make the protein hydrophobic, one can add ammonium sulfate that will interact with charged groups on the protein surface and mask the surface charges making it hydrophobic. Proteins are then eluted with a decreasing concentration gradient of ammonium sulfate-containing buffer i.e. making proteins less hydrophobic. Buffer containing a decreasing concentration of ammonium sulfate.

Question 5

Affinity chromatography: Ni-NTA chromatography

Answer 5

Proteins are separated based on the affinity of hexahistidine to Nickel ion. Protein is expressed with a hexahistidine peptide tag either in the N or C-terminus. This tag will then chelate the Nickel ion on the Ni-NTA beads. Buffer containing increasing concentration of imidazole. Imidazole has the same structure as the histidine side chain and can effectively compete with the His-tag for Nickel ions in the Ni-NTA beads.

Question 6

Affinity chromatography: Glutathione matrix column

Answer 6

Proteins are separated based on the affinity of glutathione S-transferase to glutathione. Protein is expressed with a glutathione S-transferase tag usually in the N-terminus. This tag will then bind to beads containing covalently linked glutathione. Buffer containing glutathione.

Question 7

Affinity chromatography: Immobilized antibodies

Answer 7

Proteins are separated based on the affinity of antibodies to specific proteins. Antibodies that can bind specifically to the protein of interest is covalently linked to an inert matrix eg. agarose beads. These antibodies will then specifically bind to the target protein. Different pH buffer (change surface charge of protein to weaken interaction with antibody).