Purification of protein

Question 1

What are the main objectives of protein purification techniques?

Answer 1

• To isolate one particular protein from the other protein so that the structure and functions can be studied
• To increase the stability of the protein
• To produce the protein on a larger scale for commercial purposes

Question 2

List the protein purification techniques

Answer 2

1) Ammonium sulfate precipitation
2) Dialysis
3) Gel filtration chromatography
4) Ion exchange chromatography
5) Affinity chromatography
6) Hydrophobic interaction chromatography
7) Gel electrophoresis
8) Protein quantitation

Question 3

Ammonium Sulfate Precipitation

Answer 3

Method: Precipitating protein in water solution
Concepts: Increasing the concentration of salt will decrease the solubility of the protein in water

Question 4

Dialysis

Answer 4

Method: Separating large protein molecules from other small molecules through semipermeable membrane

Question 5

Gel filtration chromatography

Answer 5

Method: Separate proteins according to their size using gel filtration column

Concept: Gel filtration column consists of gel beads that will trap smaller molecules in its internal solvent
(same concept as dialysis, just that the solution will be flowing through the column and exit at one end)

Question 6

Ion exchange chromatography

Answer 6

Method: Separating proteins based on their overall net charge using anion/cation exchange resins

Concepts: Anion exchange resin separate -ve charged protein and filter out +ve charged protein
(involve electrostatic interaction between solute ions and fixed ions charge on stationary phase)

Question 7

Affinity chromatography

Answer 7

Method: Separating protein based on its molecular conformation using ligand

Concept: Using the function of ligands to differentiate certain protein with the other through their specific affinity

Question 8

Hydrophobic Interaction Chromatography

Answer 8

Method: Fractionated proteins by exploiting their difference in the degrees of surface hydrophobicity

Concepts: the protein solution will flow through a gel matrix (covalently hydrophobic)
- The more hydrophobic the protein, the tighter the binding

Question 9

Gel electrophoresis

Answer 9

Method: Separation of proteins through an electric field

Concept: When placed in an electric field, proteins will move towards one electrode or another

• Proteins are unfolded and covered by SDS (anionic detergent)
• Mobility of proteins depends on their molecular weight and net charge
• The greater the net charge, the faster the molecule will move

Question 10

Protein Quantitation

Answer 10

Concept: Determining the concentration of protein in a given sample depending on the nature of the protein (speed, accuracy, and sensitivity of assay)

Methods:

a) Biurret test
b) Folin-Ciocalteu (Lowry) Assay
c) Bicinchoninic Acid (BCA) Assay
d) Dye-Binding (Bradford) Assay
e) Ultraviolet Absorbance @ 260nm

Question 11

How are proteins being purified?

Answer 11

Proteins are purified from a mixture using combination of several techniques based on protein properties

Question 12

List the protein properties and their techniques

Answer 12

• Solubility (Ammonium Sulfate Precipitation)
• Molecular size (Dialysis, Gel Filtration Chromatography, Gel Electrophoresis)
• Molecular Charge (Ion exchange chromatography, Gel electrophoresis)
• Hydrophobicity (Hydrophobic interaction chromatography)
• Biological Activity/ Molecular conformation (Affinity chromatography)