Receptor theory 2

Question 1

What are the steps for a radioligand binding assay

Answer 1

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

Question 2

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

Answer 2

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

Question 3

How is degradation of a ligand prevented

Answer 3

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

Question 4

What is the purpose of uncubation

Answer 4

To keep the integrity of the ligand and receptor

Question 5

How is unbound ligand separated from the mixture

Answer 5

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

Question 6

What shape does specific binding show on the radioligand binding curve

Answer 6

Rectangular hyperbola - showing saturation

Question 7

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

Answer 7

Linear relationship