Week 2 - Studying Metabolism Flashcards
How do circularly permuted biosensors work?
- circularly permuted fluorescent proteins are cut and religated
- attached to a receptor of interested molecule
- fusion protein fluoresces when molecule binds
How can caspase activity be quantified?
- 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
How does TMRE work?
Tetra-Methyl Rhodamine Ester
- a fluorescent dye
- accumulates in mitochondria depending on its membrane potential
What are the types of fluorescence microscopy?
high content imaging
- involves taking pictures at specific intervals to create a time lapse
- to quantify dead cells
confocal imaging
- for sub-cellular localisation
How does a seahorse analyser work?
- 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
How are pH meters used to assess rate of aerobic glycolysis?
- 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
How can an oxygen electrode help determine ETC activity?
- 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
What is stable isotope tracing? Give an example.
- 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)
What does a high amount of a metabolite in a test suggest?
- 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
What are the cons of an NMR?
- not all nuclei have spin property
- large sample needed
- very expensive
- LC-MS preferred instead
What is an NMR?
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
How is HPLC used to separate molecules based on charge?
- 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
What is the advantage of a HPLC?
High Performance Liquid Chromatography
- involves less complicated molecule mixes
- so they are easier to identify
- for accurate quantification
How does a MS2 (tandem) mass spectrometry work?
- 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)
What is the process of a simple mass spectrometer?
- a high voltage is applied to charge the molecules (can be either positive or negative)
- molecules are sprayed onto a quadrupole (ion selector)
- pass to a holding cell to line them up
- flow through the time of flight tube towards the oppositely charged detector
- detector measures m/z ratio (mass and charge)
