Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

MBB363 - Genetics Data Handling > Thomson - protein:protein interactions & RNA > Flashcards

Thomson - protein:protein interactions & RNA Flashcards

You may prefer our related Brainscape-certified flashcards:
Biology 101 flashcards
1
Q

What are direct prot-prot interactions?

A
  • physically assoc
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What are indirect prot-prot interactions?

A
  • interacts w/ prot via another prot
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What are 3 common methods of detecting prot-prot interactions?

A
  • affinity purifications and co-immunoprecipitations
  • yeast-2-hybrid analysis
  • reconstitution studies using recombinantly expressed proteins
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What are the steps of a co-immunoprecipitation?

A
  • 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What can co-immunoprecipitation identify and what can it not?

A
  • can identify both direct and indirect interactions –> but CANT distinguish between them
  • can theoretically identify all co-purifying prot factors
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What do you need to carry out affinity purification?

A
  • prot of interest
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What are steps involved in affinity purification?

A
  • 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is the advantage of affinity purifications over co-immunoprecipitations?

A
  • don’t need specific Ab
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What is a pot issue w/ affinity purifications?

A
  • tag can theoretically change prot activity
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What can affinity purification identify, and what can it not?

A
  • can identify both direct and indirect interactions –> but CANT distinguish between them
  • can theoretically identify all co-purifying prot factors
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Why does affinity purification often have 2 steps?

A
  • cleaner
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What can be done after PAGE, and what does each identify?

A
  • WB –> identify specific co-purifying prots w/ Abs

- staining (silver/coomassie) –> identify all co-purifying prots by mass spec

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What is the diff between silver and coomassie staining?

A
  • silver = more sensitive (ie. low abundance)

- coomassie = stains all prots

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What are the problem w/ using yeast-2-hybrid to identify direct interactions?

A
  • high levels of false +ves

- only assessing direct interaction between 2 prots –> not seeing complete pic

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

How is yeast-2-hybrid carried out?

A
  • 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 well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

How can false +ves in yeast-2-hybrid be combatted?

A
  • can do diff reporters (back to back) –> diff combos
17
Q

What is the +ve control in yeast-2-hybrid (if included)?

A
  • 2 known interacting prots
18
Q

What is the -ve control in yeast-2-hybrid, and why are they needed?

A
  • prot of interest w/ non-interacting prots

- shows constructs aren’t autoactivating (activating in non specific manner)

19
Q

What can yeast-2-hybrid identify?

A
  • direct interactions –> assesses in a cellular context
20
Q

What is heterologous expression?

A
  • prot from 1 org expressed in diff species
21
Q

What does in vitro binding assays of recombinantly expressed prots involve?

A
  • heterologous expression of prots
  • purification
  • incubate w/ purified prot 2
  • wash and elute
  • +ve interaction if bound to bait prot, -ve if not
22
Q

What must be considered when saying something is part of the interaction?

A
  • is it necessary for interaction?

- is it sufficient for interaction?

23
Q

How can eluates of RNA-IP be analysed, and what does each method identify?

A
  • 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
24
Q

Why is a denaturing gel used in Northern blotting?

A
  • to unfold RNA –> as usually forms tight 2°/3° structures which affect migration
25
Q

What are the steps involved in northern blotting?

A
  • 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
26
Q

What are the steps in primer extension?

A
  • RNA sample = total RNA or IP RNA
  • anneal labelled probe
  • RT reaction
  • run cDNA on denaturing acrylamide gel
  • mature migrates furthest as product smallest

MBB363 - Genetics Data Handling (4 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions