Ligand Binding Flashcards
What are the qualititative methods of observing ligand binding?
Direct visualisation Genetic complementation/yeast 2 hybrid TF Fluorescence microscopy FRET Co-immunoprecipitation
What are the quantitative methods of observing ligand binding?
Biophysical radiolabel binding assays
Isothermal Calorimetry
Competition assays
What resolution can fluorescence light microscopy detect?
200nm
What distance can FRET detect?
2-8nm
What resolution is PALM/STORM microscopy?
20-100nm
Where is fluorescence microscopy used?
Light in living cells
PALM/STORM in fixed cells
Can fluorescence microscopy detect proteins?
No, resolution larger than 2-20nm proteins
Why is quantitative characteristation needed?
Eliminate non-specific binding Identify type of interaction Determine affinity of interaction Identify stability of complex Understand regulation by PTM and effects of solution
What is the main problem with qualitative methods?
Don’t determine direct/indirect interaction
Which test is the qualitative “Gold Standard”?
Co-immunoprecipitation
How can co-immunoprecipitation be made quantitative?
PKA subunits can be separated by cAMP and bound using differently tagged fluorescent antibodies
What type of kinetics must be used for pull-down assays?
Slow reaching of equilibrium/Kd
What type of affinity has a -∆G?
Small Kd/ high affinity
Outline biophysical assay process?
Label ligand and characterise radioactive signal
Incubate with cells
Wash to remove NSB
Separate bound ligand from unbound
Cell and label counting
Determine control for NSB using excess ligand
How can a biophysical assay be used to find Kd?
Plot [Bound ligand] against [bound/unbound]
slope is -1/Kd
What factors limit a biophysical assay?
Complex is unstable for washing
Difficult to separate bound/unbound
NSB masking specific binding
Low receptor concentrations
How can low receptor concentrations be assayed?
Sub-stoichiometric approximation using rearranged equations
How can a high affinity ligand be assayed?
Using a competition assay to observe [RL] as a function of [C]
Assumes [L=L0] and [C=C0],
Low [R] and low [L]
Kdapp ~ Kc
What range of Kd can be detected in a competion assay?
1pM-1nM
What is a label-free binding assay also called?
Isothermal calorimetry
What range of Kd can be detected in a binding assay?
1nM-1mM
How does ITC measure Kd?
Measures heat for ∆H
∆G= RTln(Kass)
Plot time against heat. Gradient is Kass
What must be kept constant during ITC?
State variables: T, P, V, [C]
What is the concentration dependance of ITC?
C=Ka[R]
