cAMP Binding Assay Flashcards
What are ligand binding assays used for ?
Measuring interactions between two molecules
What is the detection method used to determine the presence or extent of the ligand receptor determined by ?
Fluorescence or radioactivity detection methods
What are used to measure fluorescence intensity ?
- Fluorescence correlation spectroscopy
- Time resolved fluorescence
- Fluorescence polarisation
- Fluorescence an bioluminescence resonance energy transfer
What is the advantage of fluorescence?
Has a broad spectrum of wavelenghts therefore can apply multiple colours
What are the disadvantages of fluorescence ?
- Suscpetible to different fluorescen interferences
- Labelling of a ligand can lead to undesirable alterations in binding characteristics of the ligand
- Difficult to obtain good fluorescent ligands for many receptors
What are the advantages of radioactive binding assays ?
- Most popular/best assay for membrane -bound targets
- Good robustness, precise determination of receptor density and distribution, ligand binding sites and affinity
What are the disadvantages of radioactive binding assays ?
Potential hazards of handling low levels of radioactivity
How do we measure radioactivity ?
Liquid scintillation counting
What is a radioligand ?
The radioactivly labelled molecule that can associate with a target protein
What does radioliganf binding assays enable us to do ?
Analyse the interactions of hormones, neurotransmitters and second messengers with receptors and target proteins
Why will a radioligand associate with a receptor, transporter, enzyme or any protein of interest ?
Is chemicall and therefore biologically indistinguishable from unlabelled ligands
What does measuring the rate and extent of binding provide ?
Information on the number of binding sites, their affinity and pharmacological characteristics
What can radioligand binding structures be used to ?
- Characterise receptors in their natural environment as well as those transfected into cell lines
- Study receptor dynamics and localisation
- Identify novel chemical structures that interact with receptor
- Define ligand activity and selectivity in normal and diseased states
What are the three experimental types of radioligand binding assay ?
- Saturation assays
- Competitive assays
- Kinetic assays
What are saturation assays used for ?
Analyse the equilibrium binding of radioactively labelled ligand to the receptor, by using concentrations of ligand and a fixed receptor level
What does the saturation assay measure ?
The tissue/cell specific affinity (Kd) and the density of the analysed receptor (Bmax)
What is the competitive assay ?
Investigates equilibrium binding at a fixed concentration of radioligand and in the presence of different concentration of an unlabelled competitor
What is a hot ligand ?
Radioactively labelled ligand
What is a cold ligand
Unlabelled ligand
In a competitive assay, what does binding data analysis provide ?
The affinity of the receptor for the competitor molecule (Ki value)
What are kinetic assay ?
Used to determine receptor/ligand pair specific dissociation and association constants (Kon/Koff)
What is Bmax and its units ?
Number of binding sites
moles/mg
What is Kd ?
Equilibrium binding constant /measure of ligand affinity
What is the scatchard plot ?
This plots the ratio of specifically bound over free ligand to specifically bound
What is non-specific binding ?
Binding to receptors other than their own and to non-receptors such as carrier proteins or enzymes
How do we measure non-specific binding ?
by adding an excess of competitor to the incubation mixture so that the binding of the radioligand to the receptors is completely displaced
but care must be taken to only to dislodge from the radioligand receptor site only and not other non-specific sites
What is the recommendation for choosing a competitor to measure non-specific binding ?
Choose a potent competitor whose chemical structure is quite distinct from that of the radioligand
What are desirable properties of radioligands ?
- High affinity to favour specific over non-specific binding
- Low non-specific binding
- High specific activity to detect low receptor densities
- Receptor specificity
What is the standard method to quantify the radioactivty of low energy radioisotopes?
Liquid scintillation
What are the two basic components of LSC cocktails to efficiently transfer the emitted energy into light ?
- Aromatic, organic solvent
- The scintillators or fluors
What are the four steps in the schematic overview of scintillation process ?
- Radioactive molecule
- Solvent molecules
- Fluor molecule
- Photomultiplier tube
What is LSC measured in ?
Counts per minutes
What are the principles of LSC ?
- beta particles are emitted which cause solvent molecules to become excited
- The energy of the solvent molecules is transferred to the fluor molecules which in turn emit light
When does quenching occur ?
When energy emitted by a radioisotope is not transferred completely into light and therefore is not detected by the photomultiplier tube of the counting instrument
What are the three types of quenching ?
- Physical
- Chemical
- Colour
What is physical quenching ?
The radioisotope becomes physically separated from the solution in which the fluor is dissolved ?
How do you avoid physical quenching ?
Properly homogenising the solution
What is chemical quenching ?
The beta particle can be absorbed by the so called quenching agents which will not re-emit the energy
What is colour quenching ?
The energy transmission could occur correctly by once the light is emitted by the flour or scintillator, it may be partially blocked by colour quenchin
What happens as a result of colour quenching ?
The signal deteced at the photomultiplier tube will not represent the total quantity of light truly emitted,
What causes colour quenching ?
Coloured samples or dirty plastic scintillation vials
What are disintegrations per min (dpm) ?
Describes the rate at which the atoms in the radioisotope are decaying
What are counts per minute ?
The rate at which events are being registered by an instrument
When are count rated = to disintergations ?
If the instrument/counter is 100% efficient and the backgroun radiation is negligeble (otherwise count rate is less than disintegrations)
What type of isotopes can be counted with more efficiency ?
Those that decay with high eneergy
What is the SI unit of radioactivity ?
Becequerel
What is another measurement of radioactivity ?
Curie
What is specific activity of a radiolabelled compound ?
The amount of radioactivity per unit mass in a sample often expressed as Ci/mmol
What are the two types of strategie adopted for the saturate binding analysis to determine receptor affinity and density ?
- Increasing the amount of radioligand added while maintaining constant both specific activity and concentration of radioligand
- Decreasing the specific activity of the radioligand due to the addition of an unlabelled ligand
How do you separate bound from free ?
- Filtration
- Centrifugation
- Equilibrium dialysis
When is the filtration method used ?
Membrane bound receptors
When is the centrifugation method used ?
Soluble receptors
What issues are in equilibrium dialysis ?
- Degradation or sticking of receptor ligand
- Cumbersome
- Time needed to obtain equilibrium
What is added in the centrifugation method to mop up ?
charcoal
What is cAMP ?
A small water soluble second messenger molecules derived from ATP
What is the enzyme used to breakdown cAMP ?
Phosphodiesterase
What does the presence of non-radioactive cAMP lead to ?
- Total concentration of cAMP increases
- The specific radioactivity of the H3 cAMP decreases proportionally to the dilution of hot with cold cAMP
What does the IC50 value give you ?
An approximation of Kd
What is theophyline ?
An inhibitor of phosphodiesterase and prevents the breakdown of cAMP to 5’AMP
