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Flashcards in Receptor theory 2 Deck (7)
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What are the steps for a radioligand binding assay

1. Tissue selection - could be from cultured cells or isolated cells from an organism.
2. Creation of radioligand
3. Mixing and incubation period
4. Separation of unbound ligand from bound complexes
5.Radioactive counter used to determine how much of the ligand is bound


What is an issue with binding assays and how is it overcome

The binding of the radioactive ligand can be non specific and bind to other things within the tube
Overcome by - 2 rows of tubes
row 1 has increasing concentrations of radioligand
row 2 has increasing concentrations of radioligand and an excess of non radioactive ligand
Non-radioactive ligand outcompetes the radioactive ligand for selective binding to the receptor
take row 1 from row 2 leaves you with specific binding for the radioligand


How is degradation of a ligand prevented

1. Free radical scavenger (ethanol) in drug solution
2. Store at low (not freezing) temperatures
3. Avoid light
4. Incorporation of antioxidants and additives - protease inhibitors


What is the purpose of uncubation

To keep the integrity of the ligand and receptor


How is unbound ligand separated from the mixture

Through filtration or centrifugation - but this is time dependent as removal of unbound ligand also promotes the removal of bound ligand - The higher the affinity of the ligand for its receptor the longer it will remain bound


What shape does specific binding show on the radioligand binding curve

Rectangular hyperbola - showing saturation


What shape does non-specific binding show on the radioligand binding curve

Linear relationship