Radioligand Binding Flashcards
Does radioligand binding measure response?
No
What can iodine be used to tag in radioligand binding?
Certain amino acids on polypeptides/proteins
Why are radioligand binding experiments completed?
- Shows how quickly a drug binds and dissociates from the receptor
- Shows whether a receptor is present in a tissue
- Shows how many receptors are present
- Can be used to tell how well unlabelled drug binds to the receptor
Outline how a basic binding assay is completed
- Buffer, radioactive ligand, and cell membranes are incubated
- Pour incubation over a filter so only free drug molecules pass through
- Scintillation counter counts reactivity left on filter- producing a value for the drug which has bound to receptors
What are the 3 main types of ligand binding assay?
- Saturation
- Kinetics
- Competition/displacement
How is a saturation ligand binding assay completed?
- Run a basic binding assay
- Repeat experiment with increasing concentrations of radioligand to find total binding
- Repeat experiment but add the same high concentration of non-radioactive ligand (to find non-specific binding)
Is non-specific binding saturable?
No
What is non-specific binding?
Binding of a ligand to anything other than a receptor e.g. binding to the membrane
What shape is a non-specific binding-[radioligand] graph?
Linear
What shape is a specific binding- [radioligand] graph?
Rectangular hyperbola
What does the KD of a binding-concentration graph represent?
The concentration of radioligand at which 50% of receptor binding sites are occupied
What condition is required for the values of Bmax and KD to be valid?
Equilibrium must have been reached
Briefly evaluate saturation binding
- Expensive as it uses lots of radioligand
- The only option if only one ligand exists for a receptor
What kind of graph is produced in a kinetics binding asssay?
Binding-time graph
Is the plateau from a binding-time graph Bmax?
No, it simply shows the maximum binding of that particular concentration of radioligand
Outline how to complete an on-rate kinetics binding assay
- A single concentration of radioligand is incubated for progressively longer times, then filtered and counted (gives total binding)
- The same experiments are repeated but with addition of a high concentration of competing non-radioactive ligand (gives non-specific binding)
Outline how to find the total binding of an off-rate kinetic binding assay
- Set up incubation and leave until eqm has been reached
- Then disturb eqm by adding a very high concentration of displacing ligand in a large volume of buffer
- Radioligand dissociates and does not reassociate
- This is repeated and incubated for different amounts of time
Outline how to find the non-specific binding of a kinetic binding assay
- Same as specific binding but with the addition of a large concentration of competing non-radioactive ligand
Why are kinetic binding assays completed?
- To determine Kon, Koff, and KD
- To work out how long it takes for a new ligand to reach eqm
Why is Kon hard to determine?
Both association and disassociation can occur
Outline how to complete a competition binding assay
- Use the same [radioligand] in all tubes but increasing concentrations of displacing non-radioactive ligand, filter and count (total binding)
- To find non-specific binding use the same [radioligand] with a very high concentration of non-radioactive competing ligand
What is IC50?
The [displacer] that displaces 50% of a specific radioligand binding
What is Ki?
The KD of the displacing ligand
Is IC50 a constant?
No
