Practical 8 : Radioligand binding assay

Question 1

what does a radioligand binding assay determine

Answer 1

the binding affinity between endogenous ligands and their receptors using specific radiolabelled ligands.

Question 2

what can a radioligand binding assay be used for

Answer 2

measuring the affinity of competing ligands for a receptor- ie as a measure if how strongly a ligand binds to its receptor

Question 3

in the practical you study the binding affinity of an unlabelled ligand to mAChR by testing its ability to interfere with the binding of a labelled ligand 3H-NMS. how

Answer 3

a competitive binding assay- vary the concentration of benzilylcholine (unlabelled ligand) while the concentration of 3H-NMS remains constant

Question 4

the membrane sample, the 3H-NMS and the benzilylcholine are incubated together and are allowed to reach equilibrium. how do we separate the bound 3H-NMS from the 3H-NMS free in solution

Answer 4

by vacuum filtration

Question 5

safe handling of radioisotopes, tritium is a low energy beta emitter, what are some guidelines for handling radioactive samples

Answer 5

wear protective clothing eg gloves, coats
don’t eat smoke chew
restrict work to spill tray

Question 6

what is non specific binding

Answer 6

in addition to binding to the receptors, the 3H-NMS will also bind to other sites known as non specific sites

Question 7

how can we assess non specific binding

Answer 7

measure conc of 3H-NMS in the presence of a saturating concentration of an unlabelled drug such as methylatropine MeAtr which occupies virtually all the receptors so H3NMS can only bind to non specific sites

Question 8

how can we find the maximum number of binding sites that can be bound by the conc of 3H NMS that you use in ur assay

Answer 8

measure the amount of 3HNMS bound to [rotein after incubation with 1x10^9 3HNMS for 30 mins at room temp ie tube 1

Question 9

how can we determine the % of specific binding of benzilylcholine at any conc to displace 2hNMS from its specific binding sites

Answer 9

calculating the remaining percentage specific binding ogf2HNMS eg
specific binding in presence of drug I(BCH) ie tubes 2 - 7 /
maximum specific binding ie tube 1
x 100%

Question 10

what is te IC50

Answer 10

the molar concentration of competing ligand BCh which reduces the specific binding of a radioligand by 50% of the amount bound in the absence of competitor