Wilson - mRNA export Flashcards
1
Q
What are pull down assays a type of?
A
- affinity purification
2
Q
Where are pull down assays carried out?
A
- in vitro
3
Q
How do pull down assays work?
A
- bait protein affinity tagged, so interactions w/ other prey proteins observed via co-purification
- wash steps remove non-interacting proteins
- analyse w/ SDS PAGE
- bait protein “pulls down” prey protein from reaction
4
Q
What is the source of prey prots dep on in pull down assays?
A
- dep on whether confirming a previously suspected protein–protein interaction or identifying unknown interaction
5
Q
How is western blotting carried out?
A
- gel electrophoresis to sep prots
- transfer to membrane
- membrane blocked to prevent nonspecific binding of Abs to membrane
- probe w/ 1° then 2° Ab
- 2° Ab often complexed w/ enz that prod detectable signal when binds substrate
6
Q
What is the diff between 1° and 2° Ab in Western blots?
A
- 1° = specific to prot of interest
- 2° = specific to host species of 1° Ab
7
Q
How do you find mRNA in FISH?
A
- use oligoT, as mRNA has polyA tail
8
Q
What are synthetic lethal screens used for?
A
- iso novel mutants whose survival is dep on prot of interest
9
Q
What can be combined w/ a synthetic lethal screen, and what would be the purpose of this?
A
- colony colour assay
- visually detect mutant dep on plasmid for survival
10
Q
What does it mean if 2 genes are synthetically lethal?
A
- deletion of 1 doesn’t affect survival
- but sim inactivation is lethal
11
Q
What is immunoprecipitation used for?
A
- iso of prots for subsequent western blots and other assay techniques
12
Q
How does immunoprecipitation differ from column affinity purification?
A
- goal is to iso just enough to be able to measure by quantitative/semi-quantitative assays
