protein targeting and export 1 Flashcards
how do we mutate GFP to get other colours
mutate the beta barrel
what other fluorescent protein is there
Ds RED
what is the fusion of protein and fluorescence called
and what is the disadvantage of using chimera
chimera
proteins may not fold properly
western blotting immunofluorescence primary and direct
specific antibody with fluorescent tag binds to antigen
western blotting immunofluorescence secondary and indirect
specific antibody binds to antigen and then a second antibody with a fluorescent tag binds to the first antibody
what does the secondary antibody give
amplification step
other useful dyes
what only fluroses when bound to DNA
Dapi
dapi and hoechst stain both do what
both bind to the minor groove
emitting blue light when exposed to UV
other useful dyes
phalloidin toxin where is it from
what does it bind to
and what does it do
death cap mushroom
f-actin
can be conjugated to different fluorophores
what is the UTRs involved in
regulation
what is 3’ UTR involved in
localisation
eg beta actin has a zip code (ACACCC)
which forms a secondary structure via base pairing with UTRs
zip code binding protein (ZBP1)
what does it contain
2 RRM- rna recognistion motif
4 KH domain
2 RRM- rna recognistion motif
what does it contain
basic regions with +ve charge which interacts with -ve charge of RNA
4 KH domain
what does it bind to
binds to ss-DNA and RNA
what does ZBP1 bind to
nascent beta actin mRNA in nucleus as apart of a longer mRNP complex
what happens to mRNPs
they are actively transported through nuclear pore complex with aid from RAN GTPase
NLS and NES how do they identify where the protein needs to go
via primary structure
NLS what is it and removal
nuclear localisation signal
not removed following transport
NLS, what kind of signals
monopartite can also be bipartite
basic likn SV40 which is rich with lysine K and arginine R
NES what is it and removal
nuclear export signal
can be anywhere in the sequence
not removed following transport
NLS what kind of signal is it
rich in hydrophobic residues
leucine L
isoleucine I
