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Molecular Structures week 3 lecture 3
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- 1What does ITC stand for?Isothermal titration calorimetry
- 2What are the 4 sources of heat in an ITC experiment?1) the protein-ligand interaction
2) dilution of ligand on injection into larger volume of sample
3) dilution of macromolecule by added ligand solution
4) mechanical mixing effects - 3Which source of heat in ITC is our objective?The proetin-ligand interaction
- 4What is the "c value"A unitless parameter that determines the shape of the isotherm:
- Specific for the system
- determines the shape of the binding curve
- ideally in range 10-100 - 5The equation for the C valuec = Ka[M]n
(where Ka is the association constant, M the macromolecule concentration and n the stoichiometry) - 6Dissociation constant (Kd) equationKd = 1/Ka
- 7Both ΔG equationsΔG = ΔH-TΔS
ΔG = -RTlnKa - 8regarding ITC, what do ΔH ΔG ΔS show?H: changes in H bonding/ wvw
S: changes in hydrophobic interactions
G: binding affinity - 9Name some advantages of ITCnon-destructive, any soluble components can be studied, no labeling, no immobilization, direct measurement, wide range of affinities
- 10What are the disadvantages of ITCnot high throughput, can require a large amount of material, ineffective if no heat change results, solvent effects & confrontational changes can complicate analyses
- 11what does DFS stand for?Differential Scanning Fluorimetry
- 12Advantages of DFScheap, quick - high throughput
- 13Disadvantages of DFSdestructive, limited info, prone to false negative/positive results
- 14What does SPR stand for?Surface Plasmon Resonance
- 15What does SPR give information on?association
steady state
dissociation - 16SPR advantagesTechnique is fast and time resolved so allowing association and dissociation rates to be determined, affinity as well as stoichiometry.
high-throughput, efficient, sensitive,
uses small amounts of material, Suitable for weak binding ligand interactions - 17SPR disadvantagesExpensive, Chemical modification or antibody required to immobilize target onto the chip, Involves microfluidics and can be prone to blockage with
bubbles, dust, material in analytes. - 18What does BLI stand for?Bio-Layer Inferometry
- 19BLI advantagesTechnique is fast and time resolved so allowing association and dissociation rates to be determined, affinity as well as stoichiometry.
high-throughput, efficient, sensitive, accurate, uses small amounts of material
Suitable for weak binding ligand interactions, cheaper than for SPR and does not use microfluidics. BLI is a bit more robust - 20BLI disadvantagesSensor chips, can be re-used, but this can be problematic
Chemical modification or antibody required to immobilize target onto the chip
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- Molecular structure & interactions Fichas sobre Molecular Structures week 3 lecture 3, creado por Sophia Puliasis el 07/11/2017.
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- Sophia Puliasis
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