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PSCM PRACTICAL MCQ > P5 - RAS > Flashcards

P5 - RAS Flashcards

1
Q

what is the need for a standard lane?

A

a comparison

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2
Q

what is the need for a control pre-induction sample/lane?

A

to observe no rab11 expression

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3
Q

what is the need for control post induction sample/lane?

A

to observe no rab11 expression

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4
Q

what is the need for a pre-induction lane/sample?

A

to observe no rab11 expression - induction needed to be done

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5
Q

should rab11 be detected in post induction, post sonfication lysate sample?

A

yes

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6
Q

should rab 11 be detected in soluble sample?

A

yes

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7
Q

should rab11 be detected in insoluble sample?

A

no

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8
Q

should rab11 be detected in unbound material?

A

no

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9
Q

should rab11 be detected in wash elute?

A

no

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10
Q

what weight is Rab11?

A

~20,100kDa

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11
Q

why do you need to wear gloves when handling acrylamide?

A

it is a neurotoxin

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12
Q

why must you be careful with acetic acid?

A

stain solution contains corrosive acid vapour

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13
Q

how big is the cDNA encoding Rab11?

A

1-173 residues

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14
Q

where was cDNA encoding Rab11 inserted into plasmid?

A

between NdeI and BamHI restriction sites

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15
Q

what plasmid was Rab11 inserted into?

A

pET28a

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16
Q

What E. coli strain has been engineered to maximise expression of foreign genes?

A

BL21DE3pLysSA

17
Q

How is Rab11 expressed?

A

induction by isopropylthiogalactoside (IPTG)

18
Q

How is the success of purification monitored?

A

SDS-polyacrylamide electrophoresis

19
Q

what antibiotic is used for Rab11 expression?

A

kanamycin

20
Q

using what absorbance is cell density monitored?

A

A600

21
Q

how is cell density measured?

A

light scattering

22
Q

what happens E. coli growth during the exponential phase?

A

cells double every 20 minutes

23
Q

what measure of absorbance indicates that bacteria are in exponential phase?

A

0.6-1.0

24
Q

how long does it take bacteria to reach exponential phase?

A

approx 3 hours

25
Q

why is SSSB and DSSB added to wells?

A

glycerol - helps sinking
staining - helps observations
uniform charging

26
Q

what allows Rab11 to bind to nickel agarose?

A

affinity binding via His-tag

27
Q

what is imidazole used for?

A

a rinsing buffer to unbind His-tag from nickel and form an elute of Rab11

28
Q

why is protein purification necessary?

A

to examine its structure by crystallography

29
Q

why is lysing necessary?

A

to break bacterial cell wall and allow access to proteins

30
Q

what does SDS-lysis buffer do?

A

denatures proteins
adds uniform charge for fair travel down gel

31
Q

is SDS lysis buffer bias?

A

no, adds more charge if protein is bigger in size

32
Q

why must this experiment be kept on ice at all times?

A

to prevent oxidation, proteolytic degradation

33
Q

what can happen if proteins aggregate?

A

they can appear heavier than they are and become insoluble

PSCM PRACTICAL MCQ (7 decks)

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