Separations (lectures 9&10)

Question 1

Why do we purify biomolecules?

Answer 1

study their properties
analyse their distribution
use them commercially or medically

Question 2

What is chromatography?

Answer 2

separation of substances by slow passage over or through an absorbing material
from the greek meaning ‘colourful writing’

Question 3

How do we isolate the protein source needed for chromatography?

Answer 3

with bacteria & blood we can centrifuge the samples directly
with organs & tissues we need to break them up by homogenisation which can then be filtered & centrifuged

Question 4

What is relative centrifugal force?

Answer 4

RCF = centrifugal force / g force

‘g’ is the acceleration due to gravity

Question 5

Why do centrifuges work at different speeds?

Answer 5

the larger the object the quicker it sediments in centrifuge
the smaller the item you’re trying to centrifuge, the higher the speed you need

Question 6

What is the supernatant?

Answer 6

Whats left in the liquid after centrifugation

Question 7

What is the pellet?

Answer 7

What sinks to the bottom in the centrifuge

Question 8

What other ways can we separate proteins other than centrifugation?

Answer 8

using chromatography to exploit other properties

1) size
2) charge
3) affinity - what they like to bind to

Question 9

What are the 2 stages of chromatography

Answer 9

Mobile phase
Stationary phase
The protein we want to purify binds to the stationary phase

Question 10

What are the 2 types of chromatography

Answer 10

Thin layer chromatography

Column chromatography

Question 11

How does chromatography work?

Answer 11

it exploits the affinity for the stationary phase which may be due to:

1) Charge interactions = ion exchange chromatography
2) Hydrophobicity = reverse phase/hydrophobic interaction chromatography
3) Size = gel filtration = size exclusion chromatography

Question 12

What is eluting?

Answer 12

washing molecule out of the column

Question 13

What is the eluate?

Answer 13

sample that has been eluted

Question 14

Size exclusion chromatography

Answer 14

porous bead 
small molecules go a long way in
big ones stay outside 
large molecules move faster 
leads to separation

Question 15

Ion exchange chromatography

Answer 15

Elution by increasing salt concentration which competes for the charged interaction
Protein is rich in negatively charged residues
If the stationary charge is positive, negatively charged molecules (the protein) will bind to it
We get the protein off by adding salt which competes with the charges on the surface of the protein
Protein then binds to the salt in the mobile phase so it eluted out

Question 16

Reverse phase chromatography

Answer 16

Proteins eluted by reversing the polarity of the solution from polar to non-polar with solvents such as ethanol
Molecules stick in water & are washing off in ethanol
Generally bind the molecule you want to the stationary phase and wash the others off before eluting

Question 17

Affinity chromatography

Answer 17

You need something specific for the molecule to bind to on the stationary phase
Adding free binding partner elutes the proteins
We then do specific elution by adding a drug/ affinity molecule in a free state
•This confuses the stationary phase with the mobile phase
•The molecule will then sometimes bind to the mobile phase & be eluted out

Question 18

Requirements for IMAC - Immobilised Metal Affinity Chromatography

Answer 18

Requires recombinant protein expression
•We need genetic engineering to produce the protein in a host such as bacteria
•Engineer the gene (DNA) to include a sequence of 6-10 histidine residues, usually at the N or the C terminus of the protein - a His-Tag
Histadine has an imidazole side chain that specially binds to metals like nickel or zinc

Question 19

How does IMAC work?

Answer 19

1) The protein with the tag binds to the metal (Ni2+) on the stationary phase and the rest of the other proteins are eluted from the column
2) You then add imidazole (chemically like histidine) which competes with the histatines for the nickel
3) Nickel can no longer interact with the protein so the protein can then be eluted from the column

Question 20

What is electrophoresis?

Answer 20

Carriage of small particles/molecules by an electric field
A gel (jelly containing lots of water) is used to prevent convection currents from dispersing the molecules
Molecules thus move as a band
The gels pore size controls the migration speed of macromolecules
From the greek electro (Amber) and phoresis ‘being carried’

Question 21

Summary of Gel electrophoresis

Answer 21

1) Gel electrophoresis allows the migration of proteins towards or away from an electrode to be controlled
2) Proteins can be separated using their intrinsic charge – native PAGE
3) Denatured proteins in SDS separate according to their size – SDS-PAGE
4) Combining the 2 methods gives us a 2D PAGE able to separate many hundred or proteins in one sample