Studying Signalling Paths Flashcards
What are the 2 approaches to studying
Top down from signal to effect
Bottom up from effect to signal
What are the 4 methods used
Purified proteins
Tissue culture
Animal models
Human models
What is using purified proteins Eg to study the interaction between 2 proteins
Overexpression in a system like ecoli then purifying invitro
Is purified proteins easy to manipulate / gain molecular analysis
Yes easily manipulated eg addition of phos groups or removal to see effect
What does clean data from purified proteins mean
Controlled conditions like ph etc
What does clean data allow
Repeated multiple times easily
What is the main disad of purified proteins
Not physiological, the conditions are artificial and in vitro
What needs to be known before using purified proteins
The pathway components
Is it easy to express and purify proteins
No there are some issues with extraction eg if protein becomes insoluble hard to remove or proteins can become inactive in extraction
What is promiscuity in protein purification
When 2 proteins meet which wouldnt in vivo
How is ste5 an example of how to use purified proteins promiscuity
Can be put in with kss1 components and cause the pathway to occur if starvation
What are the 2 types of cell tissue culture
Primary - extracts from human or animal tissue
Cell lines - been immortalised to divide constantly either by Chem treatment or taken from tumour
What are the ads of cell culture
More physiological from human/animal
Clean data- still controlled conditions
Easily manipulated
Many experiments can be done (multiple data points)
What conditions needs to be kept for tissue culture
Co2 and humidity
What are the disads of tissue culture
Not fully physiological eg cell lines Chem treated
Easily infected with bacteria
Expensive
Cells can change over time
Cells might not be pure- may be diff types of cell eg muscle and blood cells
What are ads of using animal models
Cheap
Disease models already available eg ob ob
Physiological to an extent
Is data clean with animal and human models
No, conditions can’t be controlled, other pathways can interfere, compensation occurs (accommodation to conditions)
What are the disads of animal models/ Human
Ethics , not easily repeated/can’t do many experiments on one model, if using cell preps may not be pure and can change, differences between animals, shortage of volunteers
What are the ads of using human models
Truly physiological
Can get feedback on how they feel
Can select people with disease
What is the rat treated with streptozotocin modelling
Streptozotocin destroys B cells which mimics diabetes 1
Which 2 animal models are for diabetes II
Ob ob mouse
Zucker rat
What is the ob ob mouse
Makes no leptin
What is the zucker rat
Defective hypothalamic receptor for leptin so can’t respond
What are radio markers for
Detection of phosphorylation
What can be used to study effect of one protein has on path
Knock out or reporter gene
What are problems with manipulation like Trying to increase camp
Cell responds eg pde , de phosphorylate
Which 3 chemicals block pde to increase camp
Ibmx, caffeine, theophylline
How does forskolin increase camp
Activates adenylyl cyclase
What can be added instead of camp to increase its effect
Non hydrolysable analogues
Which toxin can used to increase camp
Cholera or pertussis
What is ip3 sm made from
Pip2 breakdown by Phospholipase c into ip3 and dag
How do cells convert ip3 back
Into ip2 or ip4 then back to inositol then into pip2
What blocks the ip3 pathway of recycling
Lithium
How can you track ip3 production and see if the pathway is active
Label
How are binding assays used to detect camp levels
Radio labelled camp are added which compete with body camp for binding
If more radiolabel binds it means less normal camp was in the body for competition
Other than binding assays what can measure camp
Fret fluorescence
Fluorescence resonance energy transfer
Why is fret used and not probes or dye
Can’t use probes or dye for camp
What happens when camp binds
Change of fluorescence/ emission of light
What 2 fp used and what wavelength do they emit without camp
Yellow fp
Cyan fp
Cyan absorbs and transfers light into the yellow fp and it emits light at 540nm
What happens when camp binds in camp binding domain of fret molecules
Conformation change
Light can’t transfer from cyan to yellow fp so it is emitted by cyan at 480nm instead of 540 nm
How do you track ip3
Add tritiated inositol which would radiolabel ip products by making them and then block path by lithium
How are proteins radiolabelled when detecting phos using acrylimide and photography
Add radio P onto atp and it is y phosphate so it transfers to proteins
How are proteins separated on the aceylimide
By mw (changes to heavier when phosphorylated)
What activates pkc in phos studies
Phorbol esters
What activates pka in phos studies
8 brc amp
What is a general kinase inhibitor which blocks atp binding domain = not specific
Staurosporine
What do specific kinase inhibitors bind
Substrate binding region
Which kinase inhibitor is for pkc
Wortmannin
What needs to be added to phos studies
Phosphatase inhibitors
What phosphatase is blocked by okadaic acid and vanadate
Ser/thr phosphatase
Vanadate- tyr
What is added to genes in reporter gene to measure exp levels eg in pathway
Luciferase or gfp
