Thomson - protein:protein interactions & RNA Flashcards
What are direct prot-prot interactions?
- physically assoc
What are indirect prot-prot interactions?
- interacts w/ prot via another prot
What are 3 common methods of detecting prot-prot interactions?
- affinity purifications and co-immunoprecipitations
- yeast-2-hybrid analysis
- reconstitution studies using recombinantly expressed proteins
What are the steps of a co-immunoprecipitation?
- prep extract from cell culture
- Ab specific to bait prot immobilised on beads
- wash and elute prot (wash away non-interacting prots)
- analyse eluted prots
What can co-immunoprecipitation identify and what can it not?
- can identify both direct and indirect interactions –> but CANT distinguish between them
- can theoretically identify all co-purifying prot factors
What do you need to carry out affinity purification?
- prot of interest
What are steps involved in affinity purification?
- extract prep from culture of cells expressing affinity tagged prots
- tagged prot immobilised on beads
- wash and cleave
- then do tandem (can be 2-step) affinity purification
What is the advantage of affinity purifications over co-immunoprecipitations?
- don’t need specific Ab
What is a pot issue w/ affinity purifications?
- tag can theoretically change prot activity
What can affinity purification identify, and what can it not?
- can identify both direct and indirect interactions –> but CANT distinguish between them
- can theoretically identify all co-purifying prot factors
Why does affinity purification often have 2 steps?
- cleaner
What can be done after PAGE, and what does each identify?
- WB –> identify specific co-purifying prots w/ Abs
- staining (silver/coomassie) –> identify all co-purifying prots by mass spec
What is the diff between silver and coomassie staining?
- silver = more sensitive (ie. low abundance)
- coomassie = stains all prots
What are the problem w/ using yeast-2-hybrid to identify direct interactions?
- high levels of false +ves
- only assessing direct interaction between 2 prots –> not seeing complete pic
How is yeast-2-hybrid carried out?
- transformation of yeast w/ 2 plasmids encoding:
1) prot of interest tethered to GAL4 (TF) binding dom
2) prot of interest tethered to GAL4 activation dom - transcribed and translated
- binding dom binds to GAL4 upstream activating seq
- if 2 prots interact, GAL4 AD and BD come together and allow expression of reporter gene (eg. HIS3)
- allowing growth of yeast on -HIS media
How can false +ves in yeast-2-hybrid be combatted?
- can do diff reporters (back to back) –> diff combos
What is the +ve control in yeast-2-hybrid (if included)?
- 2 known interacting prots
What is the -ve control in yeast-2-hybrid, and why are they needed?
- prot of interest w/ non-interacting prots
- shows constructs aren’t autoactivating (activating in non specific manner)
What can yeast-2-hybrid identify?
- direct interactions –> assesses in a cellular context
What is heterologous expression?
- prot from 1 org expressed in diff species
What does in vitro binding assays of recombinantly expressed prots involve?
- heterologous expression of prots
- purification
- incubate w/ purified prot 2
- wash and elute
- +ve interaction if bound to bait prot, -ve if not
What must be considered when saying something is part of the interaction?
- is it necessary for interaction?
- is it sufficient for interaction?
How can eluates of RNA-IP be analysed, and what does each method identify?
- northern analysis –> identify specific RNAs w/ probes
- qRT-PCR –> get cDNA and quantify (need be v careful to do proper controls)
- primer extension –> mapping strategy to identify 5’ ends
Why is a denaturing gel used in Northern blotting?
- to unfold RNA –> as usually forms tight 2°/3° structures which affect migration
What are the steps involved in northern blotting?
- RNA sample = total RNA or IP RNA
- run on denaturing acrylamide/urea gel or denaturing agarose gel
- RNA transferred to membrane
- membrane hybridised w/ labelled probe
What are the steps in primer extension?
- RNA sample = total RNA or IP RNA
- anneal labelled probe
- RT reaction
- run cDNA on denaturing acrylamide gel
- mature migrates furthest as product smallest
