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PSCM PRACTICAL MCQ > P6 - c'AMP > Flashcards

P6 - c'AMP Flashcards

1
Q

What are ligand binding assays used for?

A

to measure interactions between 2 molecules

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2
Q

what methods are used to detect ligand complexes?

A

fluorescence and radioactivity detection

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3
Q

what fluorescence techniques are used for ligand binding assays?

A

F intensity, F correlation microscopy, time-resolved F, F polarisation, bioluminescence resonance energy transfer

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4
Q

what is an advantage associated with fluorescence assays?

A

can apply multiple colours

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5
Q

what is known as the gold standard in assay experiments?

A

radioactive labelling

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6
Q

What is a radioligand?

A

a radioactively labelled molecule that associates with a target proteins

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7
Q

what are radioligand binding studies used for?

A

detecting receptors and looking at ligand activity in a diseased state

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8
Q

what are the 3 types of experimental radioligand binding assay?

A

saturation, competitive, kinetic

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9
Q

what is the point of a saturation assay?

A

encovers equilibrium binding of radioactively labelled ligand to receptor by increasing concentrations of ligand at a fixed receptor concentration level

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10
Q

what do saturation assays measure?

A

cell specific affinity Kd
density of receptor Bmax

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10
Q

what do saturation assays measure?

A

cell specific affinity Kd
density of receptor Bmax

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11
Q

what is the point of competitive assay?

A

encovers equilibrium binding at fixed concentration of radioligand in presence of different concentrations of unlabelled competitor

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12
Q

what does competitive assay measure?

A

affinity of receptor for competitor molecule Ki

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13
Q

what is the point of a kinetic assay?

A

determins receptor/ligand pair specific dissociation and association constants Kon/Koff

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14
Q

what is a common way to analyse saturation binding data?

A

scatchard rosenthal plot

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15
Q

what does Scatchard plot show?

A

ratio of specifically bound/ free ligand to specifically bound

16
Q

what is the slope equal to on the stachard plot?

A

-1/KD

17
Q

where is Bmax located on the scatchard plot?

A

intersection of x axis (bound)

18
Q

what does Bmax mean?

A

the number of binding sites

19
Q

how is non-specific binding measured?

A

satuartion of competitor
radioligand cannot bind to receptor and only binds to non-specific sites

20
Q

what are desirable properties of radioligands?

A

high affinity to favour specific binding over non specific (KD of 1nm or less)
low non-specific binding
high specific activity to detect low receptor densities
receptor specificity

21
Q

how is radioliagnd binding measured?

A

liquid scintillation counting LSC

22
Q

What specific cocktail is needed for LSC detection?

A

ecoscint
aromatic organic solvent
scintillator or fluors

23
Q

how does LSC work?

A

beta particles are emitted and causes solvent molecules to become excited
the energy of solvent molecules is transferred to fluor molecules which emit light

24
Q

what is quenching?

A

energy from radioisotope is not transferred into light and cannot be detected

25
Q

what is physical quenching?

A

radioisotope is separated from solution in which fluor is dissolved

26
Q

how is physical quenching avoided?

A

homogenisation

27
Q

what is chemical quenching?

A

beta particle is absorbed by quenching agents that do not re-emit energy

28
Q

what is colour quenching?

A

colour being radiated from isotope is blocked

29
Q

what does dpm describe?

A

disintegrations per minute
the rate at which atoms in a radioactive source are decaying

30
Q

what does cpm describe?

A

counts per min
the rate at which decay events are being registered by an instrument
less than dpm rate

31
Q

which isotope has higher efficiency, 125I or 3H?

A

125I ~ 70-90%
3H ~ 60%

32
Q

What is Bq and what is it equivalent to?

A

SI unit of radioactivity (Becquerel)
= 1dpm

33
Q

what is Ci?

A

Curie
measurement of radioactivity
37GBq
2.22x10^12 dpm

34
Q

how is the specificity of a radioligand reduced?

A

addition of a competitive unlabelled ligand

35
Q

how do you separate bound ligand from free ligand?

A

filtration, centrifugation, equilibrium dialysis

36
Q

what is cAMP?

A

small water soluble second messenger derived from ATP

37
Q

How is cAMP made?

A

neurotransmitter binds to GPCR
adenylate cyclase activated
ATP to cAMP is catalysed

PSCM PRACTICAL MCQ (7 decks)

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