protein interactions Flashcards
how are proteins detected?
electrophoresis or western blot
where does protein interaction happen?
in organism or in an unrelated organism system (test tube)
what are co-immunoprecipitation experiments?
they detect protein-protein interactions
how do co-immunoprecipitation experiments work?
antibodies bind with bait protein
beads take up all the bound protein and it sinks
you remove the unbound liquid
use the beads for a western blot
what does liquid chromatography do?
allows us identification of all proteins
what does mass Spectroscopy do?
show us mass to charge ratios
what do we use if there are no antibodies?
we use tags (his-tags or FLAG-tags)
what is proximity biotinylation?
use biotin ligase to mark proteins that are close by fusing bait protein to promiscuous BirA to add biotin to exposed lysines on nearby protein (biotinylation) to then separate by binding with streptavidin beads and then putting through a streptavidin column
what is flouresence?
tells us where interaction happens (FRET)
what does FRET stand for?
fluorescence resonance energy transfer
what are disadvantages of BIFC and FRET?
only two proteins at a time
time consuming
needs fluorescence microscope
what are the advantages of BIFC and FRET?
good resolution (single cell or better)
temporal resolution
what is the two-hybrid system in yeast?
GA14 protein splits into AD and BO (one on bait one on prey) and when they are together they form/activate transcription of a reporter
what is the Y2H experiment?
it detects physical protein interactions through activation of the GA14 reporter gene (to make yeast blue)
