radioligand binding assays

advantages of radioligand binding assays

• Simple

• Cheap

• Flexible

• Scalable

• Sensitive

disadvantages of radioligand binding assays

• Hazardous to work with

• Waste difficult to dispose of - due to long half life

  • expensive

incubation step

• mix the receptor and radioligand

• binding to the receptors

• will then come to an equilibrium

  • some will be bound some will remain unbound

• some radioligand may bind to membranes - NON SPECIFIC BINDING

what is non specific binding

radioligand doesnt bind to receptor, binds to membrane instead

separation for binding assays

• pass tissue sample and solution through glass fibre filter

• pores are one micrometer - allow unbound to pass through, tissue will stick to this filter

• vacuum underneath the filter will push it through

quantification

• liquid scintillation counter

  • mix tissue sample with scintillin solution, measure how much light is emitted from solution

• gamma counters

  • solid scintillin, can be reused between samples

what is liquid scintillation counter used on

beta emitting nuclides such as titrium

what is gamma counter used on

iodine-125

use of saturation binding assay

used to find Kd (affinity) and Bmax (maximum binding sites) for a specific radioligand

Kd

the concentration of drug which produces 50% binding

relationship between Kd and affinity

• as Kd increases, affinity decreases

problems with radioligand binding assays and drug screenings

• need a radioactive version of every drug that you want to investigate

• expensive - high conc needed

• time consuming to make

• impractical for drug screening