P6 - c'AMP Flashcards
What are ligand binding assays used for?
to measure interactions between 2 molecules
what methods are used to detect ligand complexes?
fluorescence and radioactivity detection
what fluorescence techniques are used for ligand binding assays?
F intensity, F correlation microscopy, time-resolved F, F polarisation, bioluminescence resonance energy transfer
what is an advantage associated with fluorescence assays?
can apply multiple colours
what is known as the gold standard in assay experiments?
radioactive labelling
What is a radioligand?
a radioactively labelled molecule that associates with a target proteins
what are radioligand binding studies used for?
detecting receptors and looking at ligand activity in a diseased state
what are the 3 types of experimental radioligand binding assay?
saturation, competitive, kinetic
what is the point of a saturation assay?
encovers equilibrium binding of radioactively labelled ligand to receptor by increasing concentrations of ligand at a fixed receptor concentration level
what do saturation assays measure?
cell specific affinity Kd
density of receptor Bmax
what do saturation assays measure?
cell specific affinity Kd
density of receptor Bmax
what is the point of competitive assay?
encovers equilibrium binding at fixed concentration of radioligand in presence of different concentrations of unlabelled competitor
what does competitive assay measure?
affinity of receptor for competitor molecule Ki
what is the point of a kinetic assay?
determins receptor/ligand pair specific dissociation and association constants Kon/Koff
what is a common way to analyse saturation binding data?
scatchard rosenthal plot
what does Scatchard plot show?
ratio of specifically bound/ free ligand to specifically bound
what is the slope equal to on the stachard plot?
-1/KD
where is Bmax located on the scatchard plot?
intersection of x axis (bound)
what does Bmax mean?
the number of binding sites
how is non-specific binding measured?
satuartion of competitor
radioligand cannot bind to receptor and only binds to non-specific sites
what are desirable properties of radioligands?
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
how is radioliagnd binding measured?
liquid scintillation counting LSC
What specific cocktail is needed for LSC detection?
ecoscint
aromatic organic solvent
scintillator or fluors
how does LSC work?
beta particles are emitted and causes solvent molecules to become excited
the energy of solvent molecules is transferred to fluor molecules which emit light
what is quenching?
energy from radioisotope is not transferred into light and cannot be detected
what is physical quenching?
radioisotope is separated from solution in which fluor is dissolved
how is physical quenching avoided?
homogenisation
what is chemical quenching?
beta particle is absorbed by quenching agents that do not re-emit energy
what is colour quenching?
colour being radiated from isotope is blocked
what does dpm describe?
disintegrations per minute
the rate at which atoms in a radioactive source are decaying
what does cpm describe?
counts per min
the rate at which decay events are being registered by an instrument
less than dpm rate
which isotope has higher efficiency, 125I or 3H?
125I ~ 70-90%
3H ~ 60%
What is Bq and what is it equivalent to?
SI unit of radioactivity (Becquerel)
= 1dpm
what is Ci?
Curie
measurement of radioactivity
37GBq
2.22x10^12 dpm
how is the specificity of a radioligand reduced?
addition of a competitive unlabelled ligand
how do you separate bound ligand from free ligand?
filtration, centrifugation, equilibrium dialysis
what is cAMP?
small water soluble second messenger derived from ATP
How is cAMP made?
neurotransmitter binds to GPCR
adenylate cyclase activated
ATP to cAMP is catalysed
