Biomolecular Interactions
Interaction assays equipment
We can analyse small molecules, peptides, proteins, DNA, RNA, sugars …The facility has SPR, ITC and MST machines.
Which method would work best to test interactions of the molecules you’re studying?
Affinity KD (molar, M) is the key measurement, describing strength of an interaction. SPR, ITC or MST will give you this number. Sometimes we need to use KA ; KD = 1 / KA.
Each technique also allows us to find additional information.
- Association rate ka (M-1 s-1) and dissociation rate kd (s-1, for a 1:1 interaction)
- Enthalpy change ΔH (kcal/mole or kJ/mole; 1 cal = 4.184 J)
- Entropy change ΔS kcal/mole/oC, kJ/mole/K
- Stoichiometry and activity
- Cooperativity and avidity effects
- Specificity - comparison of KD, ka and kd between interactions.
Strengths of each interaction analysis technology |
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|---|---|---|
SPR |
ITC |
MST |
|
KD, ka and kd |
KD, ΔH, ΔS |
KD, cooperativity |
|
kinetics |
thermodynamics |
low sample requirements |
|
high-throughput screening |
solution measurements |
easy to use |
|
small-molecule binding |
stoichiometry (or % activity) |
|
What do we need for each assay?
Across all these binding assays, we get better results with well-matched buffers and accurate concentrations of both partners. Have you got positive and negative controls for your assay?
Biacore surface plasmon resonance- SPR
In addition to equilibrium affinity (KD), Biacore gives kinetic data, the association and dissociation rates (ka, kd) for additional characterisation of the interaction.
One molecule, the “ligand” is immobilised on a chip surface and the binding partner “analyte” injected across the surface. The optical phenomenon of SPR (surface plasmon resonance) is used to monitor binding and dissociation.
Immobilisation strategies include using amine groups on the protein surface, biotin-streptavidin or capture using an anti-tag antibody. Let’s find the best strategy for your molecules.
With four flow-cells, we can analyse binding to three surfaces derivatised with different molecules (and with one reference flow-cell). This adds “specificity” information, comparing affinity and kinetics for the different interactions.
Think about controls, mutants, truncated variants, homologues or different density to use all flow-cells.
The facility has Biacore 3000 and T200 machines. Both can be programmed for unattended, overnight operation. The T200 is specially designed for small-molecule binding assays.
What do we need for each assay?
Across all these binding assays, we get better results with well-matched buffers and accurate concentrations of both partners. Have you got positive and negative controls for your assay?
Isothermal titration calorimetry – ITC
ITC measures the heat changes when two molecules interact. This allows us to monitor binding in solution (without immobilisation) and without fluorescent labelling. As there are heat changes when almost all molecules interact, ITC is very widely useful for assays of different kinds of molecules. Titrating one molecule into a solution of the other gives a series of injection spikes, representing the amount of heat change. These signals get smaller with each injection (of the same volume) showing the binding sites filling up. The facility has a Microcal ITC200 machine.
What do we need for each assay?
Across all these binding assays, we get better results with well-matched buffers and accurate concentrations of both partners. Have you got positive and negative controls for your assay?
Micro-scale thermophoresis – MST
An infra-red (IR) laser generates a small temperature gradient, of just a few degrees. Different molecules move different amounts along the gradient. Receptor and ligand-receptor complexes behave differently due to differences in size, charge and hydration. Fluorescent-labelling of one molecule allows us to quantitate the different properties of receptor versus receptor-ligand complexes. Titrating an unlabelled molecule from very low concentrations to saturating concentrations gives us a binding curve. The filters in our machine allow us to use fluorophores with Ex/Em wavelengths at 490/520nm and 550/620nm.
What do we need for each assay?
Across all these binding assays, we get better results with well-matched buffers and accurate concentrations of both partners. Have you got positive and negative controls for your assay?