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