Toolkit Techniques

Question 1

What are these used for

Answer 1

Mainly medical diagnostics

Question 2

Antibodies

Answer 2

• Complex proteins that have an extremely high affinity for target antigens
• Defend against disease
• 2 identical heavy chains
• 2 identical light chains
  2 identical antibody binding sites
  Can make millions of different antibody molecules
  Variations in antibodies has been created through a process called Generation of Diversity (GOD)

Question 3

What makes antibodies

Answer 3

WBCs - Called B- Lymphocytes

Each of these resting b cells has a different membrane bound antibody

Question 4

When and antigen binds to a B cell…

Answer 4

… it divides repeatedly and secretes soluble antibody

Question 5

How do we make an antibody to order?

Answer 5

Inject animal with antigen –> after period of time, draw blood and purify

Question 6

Monoclonal antibodies

Answer 6

Consist of one single type of antibody

• Will only attach to one thing
• more expensive
• more difficult to make

Question 7

Hybridoma cell

Answer 7

Formed experimentally by fusing a single B cell from the injected animal with a B tumour cell
Will produce many monoclonal antibodies
Secretes one single antibody

Question 8

Antibodies can be used to detect proteins

Answer 8

They are conjugated to small molecules used for identification–>these anble you to see the bound protein
e.g.
Fluorescent dyes (FITC,Cy5)–> glow a particular colour when excited with a wavelength of light
Colloidal gold particles –> can label two at once with different sized particles
Enzymes (HRP) –> especially good for chromatography

Ensures that the exact protein required is present - can check whether a protein is there or not

Question 9

4 Common physical or chemical procedures that are used to disrupt cells and tissues

Answer 9

• Ultrasound (sonication)
• Mild detergents e.g. Triton X100
• Small hole pressure (needle extraction)
• Shear stress (dounce homogeniser)

Question 10

How do you create a ‘soup’ of homogenate proteins and how do you treat it

Answer 10

Disrupt the cells- should leave most membrane bound organisms in tact
Then centrifuge to separate different parts of the homogenate. High speed centrifuges use refrigeration and low pressure changes.

Question 11

3 Common types of Centrifugation

Answer 11

1. Differential centrifugation
   - Separation on basis of size and density
   - Increasing speed seperates smaller organelles
   - Multi step procedure
2. Velocity sedimentation
   - Separation on the basis of speed through a salt/sugar solution
   - Typically a sucrose gradient for stability
   - Removal via base of tube
3. Equilibrium Sedimentation
   - Separation based on the buoyant density
   - Use of high density sucrose or caesium chloride gradient
   - Removed via the base of the tube.

Question 12

Isolating proteins

which properties do you look at

Answer 12

Size 
Shape 
Charge 
Hydrophobicity 
Affinity

Question 13

Column Chromotography

Answer 13

Used for separating proteins

Column contains a support matrix equilibrated with a solvent
The protein mixture is applied to the top of the tube
Large volume of solvent is pumped through the column
The proteins react to different extents with the matrix and the separated protein fraction are collected at the base of the column

Question 14

Electrophoresis and SDS-PAGE

Answer 14

Know how this works