Week 2 - Studying Metabolism

Question 1

How do circularly permuted biosensors work?

Answer 1

• circularly permuted fluorescent proteins are cut and religated
• attached to a receptor of interested molecule
• fusion protein fluoresces when molecule binds

Question 2

How can caspase activity be quantified?

Answer 2

• caspase 3/7 required during apoptosis
• fluorescence is attached to a peptide molecule
• caspase recognises it
• cleaves the molucle
• fluorescence is freed
• can bind to DNA and fluoresence

Question 3

How does TMRE work?

Answer 3

Tetra-Methyl Rhodamine Ester
- a fluorescent dye
- accumulates in mitochondria depending on its membrane potential

Question 4

What are the types of fluorescence microscopy?

Answer 4

high content imaging
- involves taking pictures at specific intervals to create a time lapse
- to quantify dead cells
confocal imaging
- for sub-cellular localisation

Question 5

How does a seahorse analyser work?

Answer 5

• combines both electrodes to measure both the oxygen consumption rate and the extracellular acidification rate to infer the relative glycolytic/mitochondrial ATP production
• they use multi-well formats

Question 6

How are pH meters used to assess rate of aerobic glycolysis?

Answer 6

• pH electrode used to measure acidification in cell culture
• sealing electrode with the dish will reveal rate of H+ production
• steps are repeated with a glycolysis inhibitor (2-deoxyglucose) to see difference

Question 7

How can an oxygen electrode help determine ETC activity?

Answer 7

• used to measure oxygen concentration in a cell culture dish
• sealing the electrode and dish together can reveal oxygen consumption rate
• same steps are repeated with the addition of an ETC inhibitor to see the difference

Question 8

What is stable isotope tracing? Give an example.

Answer 8

• a technique that allows the measurement of flux through a metabolic pathway
• they are chemically identical to its isotope so it makes no difference to the cells
• but the differing mass means they can be detected and quantified
  13C is more stable and not radioactive (unlike 14C)

Question 9

What does a high amount of a metabolite in a test suggest?

Answer 9

• the rate of the metabolite being formed is higher than the rate of the metabolite being used up
• pathway flux can be both higher or lower

Question 10

What are the cons of an NMR?

Answer 10

• not all nuclei have spin property
• large sample needed
• very expensive
• LC-MS preferred instead

Question 11

What is an NMR?

Answer 11

Nuclear Magnetic Resonance
- nuclei contain spin property which produces electromagnetic waves
- frequency of the signal depends on local molecular environment
- to quantify several molecules in a sample

Question 12

How is HPLC used to separate molecules based on charge?

Answer 12

• one side of the column is negatively charged, while the other other is positive
• ions with only a positive area can stick to the negative side and vice versa
• salt concentration is gradually increased to unstick the molecules

Question 13

What is the advantage of a HPLC?

Answer 13

High Performance Liquid Chromatography
- involves less complicated molecule mixes
- so they are easier to identify
- for accurate quantification

Question 14

How does a MS² (tandem) mass spectrometry work?

Answer 14

• quadrupole separates one type of a molecule to a holding cell
• then a fragmentation cell where there is a gas (typically N2) present to fragment the ions when colliding
• ions then pass through the time of flight tube
• first the most fragmented to the least (including ions that have not been fragmented at all)

Question 15

What is mass spectrometry?

Answer 15

analytical technique to detect and quantify molecules on the basis of mass and charge