Investigating Samples

Question 1

4 biophysical methods to investigate affinity, kinetics, and thermodynamics

Answer 1

1. Isothermal Titration Calorimetry (ITC)
2. Fluorescence Thermal Shift Assay (FTSA)/Differential Scanning Fluorimetry (DSF)
3. Surface Plasmon Resonance (SPR)
4. Bio-layer Interferometry (BLI)

Question 2

ITC

Answer 2

Measures heat changes to characterise the thermodyanmics of a biomolecular binding interactions.
Simple example: Guinea Pig surrounded by ice, measure metabolism by measuring the amount of ice melted.
Due to small amount of temperature changes, measure power instead

Question 3

ITC Directly Measures

Answer 3

Directly Measures Ka (Association constant), n (moles), and ΔH

Question 4

ITC Allows Determination Of

Answer 4

Allows determination of Kd (Dissociation constant), ΔS, and ΔG

Question 5

ITC Advantages and Disadvantages

Answer 5

Advantages: Non-destructive, no labeling or immobilization
Disadvantages: Not high throughput, ineffective when no heat change, can require a large amount of material

Question 6

FTSA/DSF

Answer 6

Mix protein with an organic dye and heat it. Denatured protein allows dye to bind to hydrophobic residues, causing a change in Fluorescence.

Question 7

FTSA/DSF Advantages and Disadvantages

Answer 7

Advantages: Cheap, Quick, High-Throughput
Disadvantages: Destructive, Prone to false positive and negative results, not all proteins suited to the technique

Question 8

SPR

Answer 8

An optical method to detect a ligand interacting with a target and from which rate and equilibrium binding constants can be obtained.
Polarised light shines onto the sensor chip coated with a gold layer and the reflected beam is detected. At a particular “angle of incidence”, the light photons cause the free electrons to oscillate.

Question 9

SPR Advantages and Disadvantages

Answer 9

Advantages: Fast, High-Throughput, Accurate, Can use small amounts of material, Suitable for weak binding ligand interactions
Disadvantages: Expensive, Prone to Blockage, Immobilization onto sensor chip is required, Involves microfluidics

Question 10

BLI

Answer 10

“Poor Man’s SPR”

Monitors the change in number of molecules that bind to the biosensor. Changes in optical thickness changes the interaction of light (interference pattern). Monitor the changes in wavelength of light scattered from the biosensor.

Question 11

BLI Advantages and Disadvantages

Answer 11

Advantages: Fast, High-Throughput, Accurate, Cheaper than SPR, Does not use microfluidics (unlike SPR), Can use small amounts of material, Suitable for weak binding ligand interactions
Disadvantages: Immobilization onto sensor chip is required, Sensor chips can be reused which can be problematic