Practicals

Question 1

How is size exclusion chromatography done?

Answer 1

On a gel
Proteins do not bind to the matrix, but are separated by size
Pore size is adjusted to separate proteins of different sizes

Question 2

Explain ion exchange chromatography

Answer 2

Molecules are separated according to net surface charge (isoelectric point (pl)).

Question 3

Explain what pl is

Answer 3

Isoelectric point
pH at which net charge of the protein is 0. In ion exchange chromatography, buffer conditions are determined by the protein’s pl

Question 4

What is anion exchange

Answer 4

Ion exchange resin containing positively charged functional groups to capture negatively charged proteins

Question 5

What are some common anion exchangers?

Answer 5

Quaternary ammonium
Quaternary aminoethyl
Diethylaminoethyl

Question 6

What are some common cation exchangers?

Answer 6

Carboxymethyl
Sulphopropyl
Methyl sulphonate

Question 7

Explain hydrophobic interaction chromatography

Answer 7

Hydrophobic residues on the surface of proteins interact with hydrophobic groups on a chromatographic matrix.
For this to take place, water must be removed

Question 8

How is water removed for hydrophobic interaction chromatography (HIC)?

Answer 8

High concentrations of salts, e,g ammonium sulfate
Under high salt conditions, proteins are more likely to bind to the matrix than stay in solution

Question 9

Outline affinity chromatography

Answer 9

Most powerful chromatographic method that uses a matrix cross linked to its own substrate.
One of the most used protein purification methods due to tagged proteins

Question 10

Give some common protein tags

Answer 10

His6
GST
maltose-binding protein

Question 11

What is psuedo-affinity chromatography?

Answer 11

A ligand of similar structure to a protein’s ligand is attached to the matrix.
e.g a matrix cross-linked with heparin is used to purify DNA-binding proteins as heparin has a similar structure to the deoxyribose-phosphate backbone

Question 12

Outline dye-chromatography

Answer 12

Variant of psuedo-affinity chromatography.
The ligands are formed by synthetic polycyclic dyes
Structural similarities between NADH and NADPH are used to purify enzymes which require adenylyl-containing substances

Question 13

What type of chromatography can select correctly folded proteins?

Answer 13

Affinity chromatography

Question 14

What sort of chromatography is used to purify glutamate dehydrogenase?

Answer 14

Pseudo-affinity dye chromatography

Question 15

What pH should the column be set to before adding glutamate dehydrogenase (GDH)?

Answer 15

7.0

Question 16

How can the amount of protein in each fraction be checked?

Answer 16

Bradford method:
Finding the absorbance of each fraction with bio-rad at 595nm (for GDH)