Radioligand binding Flashcards
How can drugs be radioactively labelled
13H - beta emitter - half life of 12.3 years
14C - beta emitter - half life of 5730 years
125I - gamma emitter - half life of 59.4 days
what are the purposes of radioligand binding experiments
It can tell you if a receptor is present and whether the receptor number changes after things like disease or drug treatment.
Tell you the affinity of the radioligand for the receptor.
How quickly the drug binds and dissociates from the receptor.
To detect how well non radioactively labelled drugs bind to the receptor.(completion binding)
what is non-specific binding
When there is binding to non-receptor sites
How do you set up a simple binding assay
In a test-tube you add a buffer, the radioactive ligand and the biological sample you want to test, (can be cell fragments, tissues or whole cells)
this forms an incubation
the incubation is now passed through filter paper under suction which allows the free/unbound ligands to pass through but not the ligand bound to receptors on the biological sample.
scintillation counting is used to count the radioactivity on the filter paper.
What are the 3 main types of ligand binding assays.
Saturation
Displacement ( completion)
Kinetics
Explain the process of saturation analysis and what information can you collect from it.
A series of incubations is produced with an increasing concentration of radioactive ligand in each tube. these are allowed to reach equilibrium and them are filtered and counted.
This gives you the count for total binding.
Make another series of incubations identical to the first but add a high concentration of non-radioactive competing ligand to each tube. then allow it to reach equ and then filter and count.
This gives you a count for non specific binding.
total binding - non specific binding = specific binding
from the specific binding and concentration graph you can find
Bmax = moles.mg protein-1 - when the curve flattens out
Kd = molar - the amount needed to occupy 50% of the receptors
