Molecular Seperations

Question 1

Why do we purify molecules

Answer 1

Study properties
Analyse the distribution in body
Use them eg for medical purpose

Question 2

What are the 2 techniques used to seperate molecules

Answer 2

Chromatography

Gel electrophoresis

Question 3

How do you acctually prepare intracellular proteins before using chromatography

Answer 3

Using a centrifuge you isolate proteins from the rest of the cell debris eg membrane

Spin at high speeds and the pellet will be cell debris, the supernatant is the proteins/molecules you want to seperate fully

Question 4

What is g force in terms of centrifugation / ultracentrifugation

Answer 4

Acceleration due to gravity (speed = centrifugal force)

Question 5

What are 4 properties chromatography exploits to seperate proteins

Answer 5

Charges

Size/shape

Affinity (binding site)

Hydrophobicity

Question 6

What is the mobile and stationary phase meaning

Answer 6

Mobile phase is the phase in which molecules move (water usually)

Stationary phase is the phase in which molecules don’t move (eg column or paper)

When molecules are in chromatography they pass the stationary phase and those that bind don’t stay mobile phase. Proteins/molecules that keep moving down are in the mobile phase

Question 7

What are the 2 forms of chromatography

Answer 7

Thin layer chromatography- using paper

Column chromatography- much longer mobile phase - seperate at a bigger scale.

Question 8

How do molecules move up in TL chromatography

Answer 8

Molecules soluble in the Eg water are part of solvent and move up with it as a solvent

Question 9

What are the molecules/proteins called when they come out of the column (don’t bind to stationary phase)

Answer 9

The eluate

The protein is eluting down the column

Question 10

How does size exclusion chromatography seperate proteins (gel filtration)

Answer 10

Porous bead are added to the column
Small molecules move into the beads but larger molecules pass by

Larger molecules = move faster (elute)

Question 11

What is chromatography which used charges on proteins to seperate called and how does it work

Answer 11

Ion exchange chromatography

If the stationary phase is +ve , proteins with a negative net charge (side chains) will bind to stationary phase

-ve proteins therefore elute

Question 12

How do you overcome ion exchange chromatography to get the protein you want

Answer 12

Add salt (naCl-) which means cl will compete to bind to stationary phase ,

The proteins then elute

Question 13

Explain the reverse phase chromatography process (using hydrophobicity)

Answer 13

Proteins with hydrophobic side chains will bind the the surface due to water (polar). This means proteins that aren’t hydrophobic will elute

Question 14

How do you overcome hydrophobic proteins sticking to the stationary phase

Answer 14

Use ethanol non polar solvent instead

Question 15

How does affinity binding chromatography work

Answer 15

Molecules with specific binding sites such as antibodies will bind to the stationary phase if there are beads attached to the complementary binding site

They therefore stay in the column

Question 16

How do you overcome affinity binding chromatography

Answer 16

Add free binding proteins/molecules to bind to the molecules instead of the beads , this allows them to elute

Question 17

What does IMac stand for and what is it used for

Answer 17

Immobilised metal affinity chromatography.

It is used for proteins that are harder to seperate

Question 18

How are recombinant proteins used in IMAC to seperate proteins

Answer 18

The proteins are engineered to have added 6-8 histidine residues(bind with metal)

This allows binding to metal such as zinc++ on the column

Question 19

How is iMac proteins eluted when they’ve bonded with metals

Answer 19

Add imidazole (structure like histidine) which allows histidine to detach and elute the protein

Question 20

How is gel electrophoresis different to chromatography

Answer 20

No mobile phase- the liquid is in gel
A gel is used to prevent convection currents (no water circling)

The gel allows movement as bands

Question 21

What controls the speed of molecules in the gel

Answer 21

Pore size of gels. There are usually different sized to stop small molecules moving too fast

Question 22

What is induced to allow movement in gel electrophoresis

Answer 22

Electric fields. There will be either or both positive or negative electrodes

Question 23

What is PAGE

Answer 23

Polyacrylimide gel electrophoresis

Question 24

What does NATIVE PAGE mean

Answer 24

Proteins seperated by size shape and charge

Question 25

Why are proteins all usually kept at ph 8

Answer 25

so that all proteins have a -ve net charge (lose protons)

This allows for the movement in one direction

Also any higher would denature the proteins

Question 26

Explain what SDS page is and how it works

Answer 26

SDS molecule is added which adds a negative overall charge of all proteins

It means all proteins are seperated by size not charge

Question 27

What is added in sds page to break disulfide bonds in proteins

Answer 27

Mercapto ethanol and they’re heated

Question 28

Does smaller proteins go further in electrophoresis (sds)

Answer 28

Yes, larger proteins appear first

Question 29

How do you calculate the molecular weight from SDS page

Answer 29

Use log 10 because it’s in a log scale and then x by the scale the protein shows on = MW

Question 30

What is the gel electrophoresis called when proteins are seperated by charges at certain ph

Answer 30

Iso electric focussing

Question 31

In IEF what is the point where the protein appears in the gel

Answer 31

The isoelectric point

Where proteins negative and positive charges are equal at a certain ph

No net charge

Question 32

What is the ph gradient produced by in ief

Answer 32

Ampholytes

Question 33

2D gel electrophoresis uses both ief and SDS. Explain the process

Answer 33

First the proteins are seperated by ief using the ph gradient
Once Ip is found
They are put in an electric field with a positive electrode at the bottom

The proteins then seperate using size (smallest goes the furthest)

Question 34

Why would more proteins appear in SDS than ief

Answer 34

Some proteins have the same isoelectric point

Question 35

Why is adding ethanol to stationary phase called reverse phase hydrophobicity chromatography

Answer 35

Because it is polar so elutes the hydrophobic proteins eventually