No need of methods to calculate binding affinity as the arrangements of association are different than the one which have been considered so far. Carbon role in protein-protein association have been reported recently by our research with lot of advancement. Otherwise ask me for report on association principle and all.
There are many methods. Biophysical methods are best applied to well-purified proteins, preferably available in multi-mg quantity. Here are some methods.
- Equilibrium dialysis
- Intrinsic (mainly tryptophan) fluorescence
- 2-D protein-observe NMR
- microscale thermophoresis
- differential scanning fluorescence
- isothermal titration calorimetry
- surface plasmon resonance
- the Hummel-Dreyer method
- equilibrium ultrafiltration
- displacement of a fluorescent ligand, measured by fluorescence intensity or polarization changes
There are many methods. Biophysical methods are best applied to well-purified proteins, preferably available in multi-mg quantity. Here are some methods.
- Equilibrium dialysis
- Intrinsic (mainly tryptophan) fluorescence
- 2-D protein-observe NMR
- microscale thermophoresis
- differential scanning fluorescence
- isothermal titration calorimetry
- surface plasmon resonance
- the Hummel-Dreyer method
- equilibrium ultrafiltration
- displacement of a fluorescent ligand, measured by fluorescence intensity or polarization changes
Again, I agree with Dr Shapiro, but like to add some protein specific issues. We have been measuring affinities using binding kinetics, which is the method of choice if the binding site of your protein is NOT readily available, as it is for the proteins of major histocompatibility class II. Equilibrium dialysis would give you erroneous results, because MHC II molecules have flexible binding grooves that is most of the time occupied with a peptide, hence the binding requires dissociation of the prebound peptide first. Please see our publication for how to kinetic measurements for MHC II binding to peptide using size separation.Methods Mol Biol. 2019;1988:343-355. doi: 10.1007/978-1-4939-9450-2_24.
good luck.
Good question Venkata Vamsi Bharadwaj Yallapragada I will also learn from the provided answers.
Over twenty years ago I used the surface plasmon resonance system of Pharmacia that gives also the real time kinetics of the binding of proteins to their substrate
Hi Venkata, though your question is too general (you can rely on Adam's answer and minimum can double the available methods), so it would be nice to know the available possibilities you have access to, nevertheless, for protein-protein interaction I would choose isothermal titration calorimetry, surface plasmon resonance, or microscale thermophoresis as a kind of standard physical methods. The rest needs more specific knowledge and could have more limitations. Even ITC and SPR are not trivial, you might need some expert assistance. Best: Karoly
First is the knowledge of the molecules and mechanism of the proteins you are taking in your study. Bit more about thermodynamics, ligand binding and kinetics and dissociation constants,concept and binding driving forces.
Methods:
You can use Calorimetric methods, biosensors and physical resonance etc.
Hi,
Surface Plasmon Resonance (SPR) and Bio-layer Interferometry (BLI) are the most commonly and popularly used technologies to analyze protein interaction. The following compares the differences in SPR, BLI and traditional techniques GST pull-down and Yeast Two-Hybrid in protein interaction study.
Characters of SPR system
Principle: SPR biosensors respond in real-time to changes in the refractive index resulting from the binding and subsequent separation of two proteins. It requires neither radiochemical nor fluorescent labels.
- High Throughput
- High Sensitivity
- Both Qualitative and Quantitative Analysis
- Low Requirement for Solution Buffer
Characters of BLI System:
Principle: The optical analytical technique analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip, and an internal reference layer. It is a label-free technique.
- High Throughput
- High Sensitivity
- Both Qualitative and Quantitative Analysis
- Low Requirement for Solution Buffer
Hope it helps!
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