Fluorescence Polarization

Question 1

How FP

Answer 1

Excitation light first time polarized
Passes through sample
Measure resulting fluorescent signal

Small tracer: low FP
Protein bound tracer (larger): high FP

Question 2

FP is dependant on

Answer 2

-molecule size
-solvent viscosity
-temperature

Question 3

Lifetime of probe

Answer 3

How long fluorescence stays in excited state
Also increases when bound to protein

Question 4

Graphs FP

Answer 4

Binding curve
Inhibition of protein (log)

Question 5

What you get FP

Answer 5

• Binding affinity KD
• Inhibition constant Ki
• Inhibition concentration at 50% inhibition IC50