Lecture 11 and 12 - Biochemical methods.

Question 1

Stages in protein purification

Answer 1

1 - Sample homogenisation causing physical disruption of cells or tissues
2 - Differential centrifugation to isolate different cellular components
3 - Separation of proteins based on solubility, size, charge, hydrophobicity, and ligand affinity

Question 2

Protein precipitation

Answer 2

Proteins can be precipitated by adding competing solutes such as ammonium sulfate ((NH₄)₂SO₄), acetone, or polyethylene glycol

Question 3

Competing solutes: what characteristics should they ideally have?

Answer 3

Very soluble in water
Relatively non-denaturing
Easy to remove
Not too viscous/dense
Cheap and pure

Question 4

Chromatography

Answer 4

Stationary phase - pigments at the bottom of the paper

Mobile phase - pigments move up the paper

Question 5

Techniques for cell disruption

Answer 5

• Mechanical - blender blades disperse cells/tissues
• Liquid homogenisation - sheared through a narrow space by a homogeniser
• Sonication - high frequency and waves shear cells using a sonicator
• Freeze/thaw - ice crystal formation after continuous freezing through a freezer disrupts cells
• Manual grinding - Grinding through a mortar and pestle

Question 6

What determines whether passive transport occurs?

Answer 6

Free energy of transport (ΔG)

If the value is positive, energy is needed to make movement occur

If the value is negative, energy is not needed to make movement occur (passive)

Question 7

Affinity chromatography

Answer 7

Immobilised molecules with affinity for a protein are used to trap a protein and then, after the rest of the solution flows ahead, a competitor molecule elutes the protein, allowing the collection

Question 8

Elution

Answer 8

The removal of a substance (usually in chromatography)

Usually by competition but can also be done by changing buffer conditions

Question 9

Types of affinity chromatography

Answer 9

Immunoaffinity chromatography
Immobilised ligand chromatography
Lectin-based affinity chromatography
Immobilised metal affinity chromatography

Question 10

Immunoaffinity chromatography

Answer 10

The antibody against protein of interest

Question 11

Immobilised ligand chromatography

Answer 11

Substrate analogue or inhibitor binds enzyme or protein (e.g. heparin and heparin-binding proteins)

Question 12

Lectin-based affinity chromatography

Answer 12

Lectins bind glycosylated proteins; elution is via the addition of sugars (e.g. N-acetyl glucosamine)

Question 13

Immobilised metal affinity chromatography

Answer 13

metal ions (e.g. Ni2+) bind engineered recombinant proteins containing a poly-histidine tag at the N- or C-terminus.

Question 14

Fusion proteins

Answer 14

Chimeric proteins engineered by the joining on the same polypeptide chain the products of two genes

Used for a variety of reasons, including affinity chromatography

Question 15

PAGE

Answer 15

Polyacrylamide gel electrophoresis

Used to separate macromolecules

Question 16

What can metal3+ elements do?

Answer 16

Bind phosphorylated proteins

Question 17

SDS-PAGE

Answer 17

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (CH3-(CH2)11-SO4–NA+)

This molecule is a hydrophobic, dual ionic character detergent molecule

It forms complexes with denatured proteins at neutral pH and can be used to detect molecular weight

Question 18

GST-fusion affinity chormatography

Answer 18

Glutathione S-transferase (GST)-fusion purified using immobilised glutathione GSH

Question 19

What do micelles do in an aqueous solution

Answer 19

Behave as phospholipids would, forming a hydrophilic outer layer and hydrophobic inner layer

Question 20

Assessment of sample purity

Answer 20

Purity - the more bands, the less pure
Abundance - the higher the band intensity, the higher the abundance

(Can only see marks after staining the gel with dye)

Question 21

Protein purification table

Answer 21

Purification level indicated by the specific activity

Question 22

What happens when proteins are at high/low pH

Answer 22

At high, the protein acts as an acid and becomes deprotonated

At low, the protein acts as a base and becomes protonated

Question 23

Isoelectric point

Answer 23

The point at which a protein has an equal number of positive and negative charges

Question 24

Isoelectric focusing

Answer 24

By creating a column with varying pH, proteins will naturally associate in the area where their isoelectric point is

Question 25

Mass spectrometry

Answer 25

Are you being fr.

The molecule gets ionized and then fragmented. Fragments are put in locations based on their weight which is gathered through the use of a magnetic field

Question 26

Polyclonal antibodies

Answer 26

Mixed populations of different antibodies which recognise different epitopes on the antigen

Question 27

Monoclonal antibodies

Answer 27

Uniform population of antibodies which recognise the same epitope on the antigen

Question 28

Immunoaffinity chromatography

Answer 28

Protein purification:
Monoclonal antibodies against the protein of interest

Adsorption:
Highly specific in favourable cases can be a one-step purification

Elution:
Tight binding can be drastic, often via pH change

Question 29

Immunoblotting

Answer 29

?

Question 30

Immunofluorescence

Answer 30

Protein labelled with a fluorescent molecule and then visualised with a fluorescence microscope

Actin filaments - red fluorescent protein (RFP)
Microtubules - green fluorescent protein (GFP)
Nuclei - blue fluorescent dye (DAPI) binding to DNA

Question 31

Immunogold labelling

Answer 31

Antibody labelled with gold colloid particle and then visualised with an electron microscope

Question 32

Passive transport across a membrane equation

Answer 32

ΔGₜ = RT x ln [substrate]ₒᵤₜ/[substrate]ᵢₙ

Question 33

Centrifugal separation equation

Answer 33

a = ω2r