Visualising gene expression and protein localisation

Question 1

How can protein localisation be studied?

Answer 1

• Antibodies

- Fusion with fluorescent protein

Question 2

How can gene expression patterns be studied?

Answer 2

• Localisation of RNA through in situ hybridisation or fluorescent RNA probes
• Promotor/enhancer studies to create a fusion transgene or promotor-trap

Question 3

What is promotor trapping?

Answer 3

• Insert reporter gene into genome randomly
• In some instances reporter will come under the influence of a promotor (trapped)
  reporter can be LacZ or GFP

Question 4

What is beta-Gal staining?

Answer 4

• Enzymatic assay
• Promotor fused to LacZ which encodes beta-galactosidase
• This uses X-gal as a subtrate to create a blue dye visualised through microscopy

Question 5

What is antibody staining?

Answer 5

• Detection of a specific antigen by an antigen (Immunoglobulin IgG) visualised by being fused with an enzyme or fluorescent dye
• May be monoclonal or polyclonal

Question 6

How can an anitbody stain be amplified?

Answer 6

• Use of a secondary antibody which recognises the primary antibody
• Species in which it was raised
• Increases number of bound signals

Question 7

What are the 5 steps of visualising tissue?

Answer 7

1. Dissect tissue
2. Fix tissue
3. Permeabilise tissue
4. Stain tissue
5. Image tissue

Question 8

What is the purpose of fixation?

Answer 8

• Denaturing the proteins so that they stay where they are
• Can be physical (heat)
• Chemical (creating covalent bonds, application of aldehydes)
• Precipitation - removing water by adding a solvent

Question 9

What is the purpose of permeabilisation?

Answer 9

• Mild surfactants lower surface tension in lipids, making holes in the membrane
• Organic solvents dissolve lipids

Question 10

What is the purpose of blocking?

Answer 10

Remove non-specific background from non-specific antibody binding (ionic interactions, van der waals forces)

Question 11

How can blocking be carried out?

Answer 11

• Application of bovine serum albumin (BSA) which competes for unspecific binding sites
• Use of mild detergent which reduces hydrophobic interactions
• High salt concentrations reduce protein-protein interactions

Question 12

What are fluorophores?

Answer 12

• Compounds/proteins which can emit light upon excitation

- Typically contain several combined aromatic groups

Question 13

What is photobleaching and how can it be reduced?

Answer 13

Reactive oxygen species destroying fluorophores

• Reduce intensity of light and exposure time
• Mounting medium which has anti-photobleaching properties (quench radicals)