Lecture 3

Question 1

After homogenisation and clarification, what’s the next step in protein purification?

Answer 1

Chromatography.

Question 2

What is the etymology for chromatography, and how was it discovered?

Answer 2

From greek - chroma = colour.
graphein = to write.
Mikhail Tsvet (1900) who invented chromatography found chlorophylls green, carotenes orange, xanthophylls yellow while analysing plant pigments.

Question 3

What solvents are commonly used in TLC (thin layer chromatography) for protein analysis? And in what ratio?

Answer 3

pet.ether : acetone (9:1).

Question 4

What is the distribution coefficient?

Answer 4

A measure of quantifying the difference between mobile and stationary phases.

Question 5

What is protein column chromatography?

Answer 5

Separation of proteins by virtue of differences in distribution coefficient of sample components between 2 different phases.

Question 6

What are the stationary and mobile phases in protein column chromatography?

Answer 6

Stationary = column matrix (gel).
Mobile = eluant (fluid used to elute (remove) a substance).

Question 7

How does protein colour chromatography work?

Answer 7

Sample loaded into column containing stationary phase. Sample starts to travel through column at different rates depending on Kd. Then solvent added and components collected separately, separated by rate of elution.

Question 8

What 4 properties of a protein can be used to help separate them? And how?

Answer 8

Size - Size exclusion chromatography.
Charge - Ion exchange chromatography.
Hydrophobicity - Hydrophobic interaction chromatography.
Biorecognition (ligand specificity) - Affinity chromatography.