lecture 2 - purification by precipitation

Question 1

What contaminants does protein removal remove?

Answer 1

• particulates
• nucleic acids
• small molecules
• contaminating proteins from the expression host

Question 2

Why is filtration not used to remove nucleic acids?

Answer 2

gelatinous/ clogs pores

Question 3

What are the pros and cons of removing nucleic acids with protamine sulphate?

Answer 3

pro - quick

con - add contaminant, poor reproducibility , adding another peptide

Question 4

What are the pros and cons of using DNase to remove nucleic acids?

Answer 4

• simple , reproducible

cons - slow, protease may degrade target

Question 5

What is the best method for removing nucleic acids?

Answer 5

sanitation ( shears chromosomes)

Question 6

How do you remove cell debris?

Answer 6

sedimentation viA centrifuging. Do not use filtration as it lowers the protein yield, as pores get clogged up with protein

Question 7

How do you remove small molecules?

Answer 7

• buffer change
• dialysis, lets out small molecules ad not proteins. keep replacing the buffer on the outside, to get rid of all the small molecules
• ultrafiltration

Question 8

What are the 5 properties of proteins that should be known so it can be isolated?

Answer 8

• Charge
• Hydrophobicity
• Affinity
• Solubility/ stability
• Molecular Weight

CHASM

Question 9

What does ‘salting in’ do?

Answer 9

• increases ionic strength from below physiological

- stabilise the proteins and increases the solubility

Question 10

What does ‘salting out’ do?

Answer 10

• removes water shielding from around the molecule by increasing the salt alot
• hydrophobic patches exposed on the protein
• promotes protein aggregation.
• ppt

Question 11

How is salting out done in practice?

Answer 11

Add salt, centrifuge and separate ppt
Increase salt, ppt protein of interest
Remaining proteins in soluble fraction

Question 12

What salt must be used in salting out?

Answer 12

• soluble , non - exothermic , pure, cheap and non interacting

Question 13

What are the pros and cons of salting out?

Answer 13

pros -Limits bacterial growth
Prevents denaturation
Concentration step

cons - Need to desalt
Ineffectual if [protein] too low
Results vary with extract components
(co-precipitation)

Question 14

What is the formula for precipitin by changing solvent?

Answer 14

F = q1 q2/ker^2

e= meant to represent the dielectric constant

r is the separation

k is a constant

Question 15

What happens when you add organic solvents to a solution?

Answer 15

• organic solvent lies over the hydrophobic sections
• makes these parts very solvated
• lowers the dielectric constant but increases electrostatic attraction causing ppt

Question 16

What is a problem with precipitation by adding organic solvent?

Answer 16

organic solvent may inactivate the protein

Question 17

What happens when a protein has a pH that is similar to its pI/ IEP?

Answer 17

A protein will be uncharged and may aggregate due to hydrophobic attraction

Question 18

What does exploiting stability do?

Answer 18

• purification by selective denaturation

- contaminants only

Question 19

How can you precipitate extremophiles?

Answer 19

denaturation by temp , pH and solvent

Question 20

What are the 5 key components of chromatography equipment?

Answer 20

1 - stationary phase 
2 - chromatographic bed 
3 - mobile phase 
4 - delivery system 
5 - detection system

Question 21

What is the stationary phase made up of?

Answer 21

a matrix - stable , inert , high capacity

Question 22

What are 4 key features of High Performance Liquid Chromatography?

Answer 22

• Incompressible stationary phases
• Pumps (High Pressure and Flow Pressure FPLC)
• Rapid separation
• Continuous monitoring

Question 23

What is the chromatography resolution equation?

Answer 23

R = separation/ average width

R = 2S / W1+W2

Question 24

What do different peaks represent on a chromatography graph?

Answer 24

unbound comes off earliest , is the first peak

Question 25

How does isoelectric precipitation by changing the ph work?

Answer 25

At a pH close to its pI , a protein will be uncharge and may aggregate , due to its hydrophobic attraction. This is isoelectric precipitation

Question 26

What limits the resolution of chromatography peaks?

Answer 26

• The peaks widen due to diffusion and retention on the column

Question 27

What salt is used for salting out?

Answer 27

(NH4)2SO4 saturated at 4M

Question 28

What is an overview of protein purification?

Answer 28

• preparation, extraction and clarification
• Capture
• Intermediate purification
• Polishing

Question 29

What does the clarification stage do?

Answer 29

Removes cells debris and other particulates

Question 30

What are the techniques used for clarification?

Answer 30

• filtration ( can lower protein yield)

- Sedimentation ( iva centrifugation on a large or small scale )

Question 31

What are the 3 ways that solubility can be exploited to purify by precipitation?

Answer 31

1. ) Changing the ionic strength ( salting in and salting out)
2. )changing the solvent
3. ) changing the pH

Question 32

What does S represent in the chromatography equation?

Answer 32

S Is the separation of the maxima

Question 33

What does W1 and W2 represent in the chromatography equation?

Answer 33

W represents the widths