lec 2

Question 1

To measure binding constant

Answer 1

find some change in property of a ligand when it’s bound to receptor

• change in absorbance
• change in fluorescence( good for dna, but only about 10% of ligands fluoresce)

Question 2

equilibrium dialysis

Answer 2

have receptor in bag, ligand outside, ligand flows in and you can measure the concentration inside and outside

Question 3

isothermal titration calomitry

Answer 3

• measures ∆H directly
• two chambers should have same temp, so when receptor binds ligand the machine measures how much heat it must add to the other chamber to make the chambers the same

Question 4

A problem with isothermal titration calomitry

Answer 4

even if theres no ligand there may be a concentration dependant heat effect when diluting the protein

Question 5

plate readers

Answer 5

for measuring Kd
advantages - measure 96 wells at once(96 assays), very rapid
disadvantages - have to link enzyme to antibody of YFP

Question 6

surface plasma resonance

Answer 6

change in angle 1 - angle 2 is converted into repsonse units, when the ligand is bound to the protein on the gold the angle changes and physics shows you that response units is proportional to the mass

• measure response units against time and flow buffer across the gold
• the response units saturate, when you change to buffer alone the curve will drop and response units will be the same so then you can get the rate constant (k1) for binding of you favourite protein