BGM1002/L08 Protein Putification

Question 1

Why are biomolecules purified? (4)

Answer 1

Study properties
Study structure
Analyse distribution and abundance
Use commercially or medically

Question 2

How are proteins studied? (2)

Answer 2

In native state
Heterozygous expression

Question 3

Give a disadvantage of using heterozygous expression to study proteins.

Answer 3

Post-translational modifications can occur in different vectors

Question 4

Give 3 biophysical properties of proteins.

Answer 4

Size
Mass
Shape/structure
Interactions
Redox potential
Charge
Hydrophobicity
Electromagnetic properties

Question 5

Give 2 ways of separating proteins.

Answer 5

Chromatography
Gel electrophoresis

Question 6

Define chromatography.

Answer 6

Separation of substances through differential interaction between two different phases

Question 7

Define gel electrophoresis.

Answer 7

Movement of small particles/molecules within an electric field

Question 8

How can proteins be isolated?

Answer 8

Centrifugion

Question 9

How does centrifugion separate proteins?

Answer 9

Based on density

Question 10

Name the 2 products of centrifugion.

Answer 10

Supernatant
Pellet

Question 11

What is centrifugal force?

Answer 11

Force appearing to act on an object when viewed in a rotating frame of reference

Question 12

What does osmotic shock do to cells?

Answer 12

Causes lysis and exposes organelles

Question 13

What is the relationship between the size of an object and the speed at which it sediments in a centrifuge?

Answer 13

Larger objects sediment more quickly

Question 14

What g force is required to prepare proteins?

Answer 14

100,000g

Question 15

Describe how to separate the supernatant and pellet.

Answer 15

Filter supernatant and dispose of pellet

Question 16

Why does chromatography work for proteins?

Answer 16

Proteins have differential interaction strength between mobile and stationary phase

Question 17

In biological chromatography, what are the stationary and mobile phases?

Answer 17

Stationary - polymer bead
Mobile - biologically compatible buffer system

Question 18

Name 3 types of chromatography.

Answer 18

Affinity
Ion exchange
Size exclusion
Hydrophobic interaction
Reversed phase
Multimodal chromatography

Question 19

What is the term for when molecules leave the chromatography column?

Answer 19

Eluding

Question 20

What kind of solvent is used in reversed phase chromatography?

Answer 20

Organic solvent

Question 21

How are molecules separated in size exclusion chromatography?

Answer 21

Hydrodynamic radius

Question 22

How are substances separated in ion exchange chromatography? (3)

Answer 22

Overall charge
Charge distribution
Charge density

Question 23

What property must the buffer have in ion exchange chromatography?

Answer 23

pH slightly higher or lower than solution

Question 24

What does high salt concentration favour in hydrophobicity chromatography?

Answer 24

Protein with media

Question 25

What is the most commonly used salt in chromatography?

Answer 25

Ammonium sulphate

Question 26

How is the stationary phase functionalised in affinity chromatography?

Answer 26

With a chemical/protein that binds protein of interest

Question 27

What does IMAC stand for?

Answer 27

Immobilised metal affinity chromatography

Question 28

What is a his-tag?

Answer 28

6-10 histidine residues added to a protein