Practical 8: Radioligand Binding

Question 1

What is Tritium?

Answer 1

• Hydrogen 3- atomic mass of 3

- low-energy beta emitter

Question 2

Why could tritium be dangerous?

Answer 2

it’s radioactive

Question 3

How was the homegenate of rat brain membranes prepared?

Answer 3

• rat cerebral cortices were homogenised in 50mM Na+- K+ phosphate buffer
• centrifuged at 800g for 5 minutes to remove unbroken cells, large debris and nuclei
• the supernatant taken off and centrifuged at 17,000g for 30 minutes to obtain and fraction consisting of membrane fragments that have resealed to form vesicles.
• supernatant removed and pellet resuspended in 50mM Na+/K+ phosphate buffer

Question 4

Define preparing serial dilutions

Answer 4

the process of making many sequential dilutions from a single stock solution

Question 5

If the BCh stock solution is 1x10^-4 M, how would you prepare 2x10^-5 M solution of BCh?

Answer 5

1x10^-4 M / 2x10^-5= 5

need it to be 5x less concentrated, so we make a 1 IN 5 dilution (1 pt. BCh to 4 parts Buffer)

Question 6

How is the % specific binding calculated?

Answer 6

specific binding in the presence of drug (BCh)/ Max. specific binding x100