chromatography • spectroscopy 27
Determining equilibrium affinity and stoichiometry in solution via light scattering
Sophia Kenrick and Daniel Some look at efficient automation of composition- gradient multi-angle light scattering measurements for quantifying protein- protein interactions.
Sophia Kenrick et Daniel Some analysent l’automatisation efficace des mesures de diffusion de lumière à angles multiples à gradient de composition.
Sophia Kenrick und Daniel Some untersuchen die effiziente Automatisierung der CG-MALS- Messverfahren zur Quantifizierung von Protein-Protein- Wechselwirkungen.
U
nderstanding macromolecular interactions is
essential for a wide range of applications - from uncovering the mechanisms of intracellular pathways to the development of biopharmaceuticals.
Proteins, nucleic acids and other biomolecules assemble, change conformation and form intricate networks with a variety of ligands to provide functions necessary for life.
Although numerous techniques exist for probing a single aspect of a complex interaction landscape, such as surface plasmon resonance (SPR), ELISA and functional assays, a label- free technique for observing a full range of associations is most desirable for quantifying physiologically
relevant reactions.
Composition-gradient multi-angle static light scattering (CG-MALS) is a powerful, label-free technique for quantifying macromolecular interactions, without the influence of fluorescent or radioactive tags or surface immobilization.
CG-MALS not only provides absolute characterisation of affinity and stoichiometry but also enables elucidating the effects of solvent composition, pH and small molecule cofactors on the interaction of interest.
At the crux of this technique is the power of multi-angle static light scattering to measure directly the apparent weight-average molar mass (Mw,app
solution.
Trough appropriate modeling of the relationship between Mw,app
and
composition, CG-MALS quantifies multiple interactions existing in an equilibrium solution, not simply at a single binding site or functional domain.
Tis tutorial describes the measurement of an equilibrium antibody-antigen association by CG-MALS, automated using the Wyatt Calypso system. To perform this experiment, stock solutions of each protein - human thrombin and a monoclonal anti-thrombin antibody - are loaded onto the Calypso hardware along with a reservoir of buffer.
) of macromolecules in
Te system automatically prepares and injects into a MALS detector a series of compositions of a protein
Fig. 1. Automated composition gradients created by Calypso system for determining affinity and stoichiometry of interactions present in a system of interacting antibody and antigen (thrombin).
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