P5 - RAS Flashcards
what is the need for a standard lane?
a comparison
what is the need for a control pre-induction sample/lane?
to observe no rab11 expression
what is the need for control post induction sample/lane?
to observe no rab11 expression
what is the need for a pre-induction lane/sample?
to observe no rab11 expression - induction needed to be done
should rab11 be detected in post induction, post sonfication lysate sample?
yes
should rab 11 be detected in soluble sample?
yes
should rab11 be detected in insoluble sample?
no
should rab11 be detected in unbound material?
no
should rab11 be detected in wash elute?
no
what weight is Rab11?
~20,100kDa
why do you need to wear gloves when handling acrylamide?
it is a neurotoxin
why must you be careful with acetic acid?
stain solution contains corrosive acid vapour
how big is the cDNA encoding Rab11?
1-173 residues
where was cDNA encoding Rab11 inserted into plasmid?
between NdeI and BamHI restriction sites
what plasmid was Rab11 inserted into?
pET28a
What E. coli strain has been engineered to maximise expression of foreign genes?
BL21DE3pLysSA
How is Rab11 expressed?
induction by isopropylthiogalactoside (IPTG)
How is the success of purification monitored?
SDS-polyacrylamide electrophoresis
what antibiotic is used for Rab11 expression?
kanamycin
using what absorbance is cell density monitored?
A600
how is cell density measured?
light scattering
what happens E. coli growth during the exponential phase?
cells double every 20 minutes
what measure of absorbance indicates that bacteria are in exponential phase?
0.6-1.0
how long does it take bacteria to reach exponential phase?
approx 3 hours
why is SSSB and DSSB added to wells?
glycerol - helps sinking
staining - helps observations
uniform charging
what allows Rab11 to bind to nickel agarose?
affinity binding via His-tag
what is imidazole used for?
a rinsing buffer to unbind His-tag from nickel and form an elute of Rab11
why is protein purification necessary?
to examine its structure by crystallography
why is lysing necessary?
to break bacterial cell wall and allow access to proteins
what does SDS-lysis buffer do?
denatures proteins
adds uniform charge for fair travel down gel
is SDS lysis buffer bias?
no, adds more charge if protein is bigger in size
why must this experiment be kept on ice at all times?
to prevent oxidation, proteolytic degradation
what can happen if proteins aggregate?
they can appear heavier than they are and become insoluble
