Overview

MDS enables the quantification of protein interactions in complex backgrounds, providing independent information on affinity, concentration, on-target / off-target binding, and stoichiometry. This information complements data obtained from other characterization techniques, ensuring a better understanding of a protein interaction and its influencing factors. By enabling scientists to go beyond simple studies of K_D and concentration, MDS facilitates more informed decisions, from basic research through to therapeutic development. 

Microfluidic diffusional sizing (MDS) exploits the unique properties of “flow” in microfluidic channels — specifically laminar flow, where streams can flow alongside one another with no convective mixing. MDS measures molecular size (hydrodynamic radius), and changes in size indicate binding events. 

The quantity of protein in each stream is determined using a fluorogenic dye. The combined total fluorescence is used to determine the concentration of protein in solution, and the ratio of fluorescence between the two streams used to determine the diffusion co-efficient of the protein, which in turn is used to determine the protein’s hydrodynamic radius.

MDS provides equivalent results in simple and complex backgrounds

Buffer

Human Serum

Figure 1. MDS provides equivalent affinities and sizes, of both the complex and free species in a traditional and complex background of > 90% human serum.

MDS reveals KD and stoichiometry, even with challenging targets

Monomers

Ologomers

Fibrils

Figure 2. MDS demonstrated no change in solution-phase affinities of antibody for the disordered α-synuclein monomer, oligomer and large, heterogeneous fibril. Binding stoichiometries were also determined, showing the binding epitope is less frequently exposed on the fibril form.

Workflow specification and benefits:

• Measurements performed in solution
• 25 min run time on the Fluidity One-M
• Compatible with a wide range of fluorogenic dyes
• K_D range from pM to µM
• Provides Stoichiometry
• Quick and easy to perform
• Use of complex backgrounds e.g., serum, lysate, CSF