Protein sizing

Knowledge of a protein’s size can provide insights on how it is folded, whether it has been expressed in a full length or truncated form, if it is in an active or inactive conformation, whether it is monomeric or oligomeric or indeed whether it is in complex with additional proteins or biomolecules.

The most widely used techniques for protein sizing rely on passing that protein through a matrix (for example Polyacrylamide Gel Electrophoresis (PAGE) or Size Exclusion Chromatography (SEC)). These approaches have a number of limitations - the results can be compromised if the protein interacts even weakly with the matrix, and the long measurement time can alter the equilibrium state and as a consequence what is observed may not be the state of the proteins in the original sample.

The Fluidity One overcomes these limitations by sizing proteins in solution, rapidly and without the use of a matrix. This is achieved using a microfluidic system under steady state laminar flow to measure diffusion; the hydrodynamic, or Stokes, radius of the protein is calculated from the observed rate of diffusion.

Laminar flow is a fluid behaviour easily achieved in microfluidic channels whereby adjacent streams of miscible fluids do not mix convectively. Consequently when a protein sample and analyte solution are run as laminar streams, the only way protein can migrate into the analyte stream is by diffusion – at a rate that is proportionate to its size. This is the measurement step in the Fluidity One system, and occurs in a native, unlabelled condition.

Following measurement, the streams are split and a latent, fluorogenic dye is introduced to label the protein and enable sensitive detection. Fluorescence intensity is then measured in both streams, the total of which is used to determine concentration of protein, and the ratio of which is used to determine the diffusion coefficient, and consequently the hydrodynamic radius.

Size Measurement on Fluidity One bar chart
Figure 1: Size Measurement Various proteins of different size measured on the Fluidity One: Insulin (5.7 kDa), RNAse A (13.7 kDa), beta-lactamase (18.3 kDa), ovalbumin (42.8 kDa), BSA (66.5 kDa), apoT (75.8 kDa), mAb (150 kDa), aldolase (157 kDa) and Thyroglobulin (660 kDa)


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The Fluidity One

In-solution sizing and quantification of native protein in 5 minutes

Fluidity One in use