Applications

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)

 

Learn more about the Fluidity One

  • Publications and resources

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    • Application note

      Protein Size as an Indication of Structure

      Molecular weight (Mw) is a commonly used, and for many scientists a readily understood, parameter to describe the size of a protein or complex. Here we show how hydrodynamic radius (Rh) can be used in combination with Mw to provide insights into the shape and structure of proteins and illustrate how Mw alone may not always provide a complete picture.

    • Brochure

      Fluidity One brochure

      The Fluidity One brochure gives an overview of the capabilities, applications and full technical specifications.

    • Application note

      Oligomerization of Interleukin-2

      A commercially available human interleukin-2 is assessed by microfluidic diffusional sizing on the Fluidity One across a dilution series. The hydrodynamic radius is observed to increase with increasing concentration, in a way which suggests a monomer-trimer equilibrium with positive cooperativity is established.

    • Blog

      What biophysical attributes of biopharmaceuticals are measured, and why?

      An array of characteristics must be investigated in new biopharmaceuticals to evaluate their safety, efficacy and behaviour under changing conditions. Here we look at what specific attributes should be measured and why.

    • Publication

      Cooperative Assembly of Hsp70 Subdomain Clusters

      Wright et al use Microfluidic Diffusional Sizing to probe the oligomerisation of the SBD641 substrate of human Hsp70. The Fluidity One was employed to verify if the fluorescent label used had an affect on measured hydrodynamic radius during the tests.

    • Blog

      Protein size - how do I measure it, and why is it important?

      An overview of why protein size matters, and what structural and functional information protein size can reveal. To understand proteins and their function, we have to understand the way they fold, aggregate and interact. Conformation is key to protein function and can be revealed by measuring size. Different methods for measuring protein size are summarised, and comparison is made, considering the method, range, cost and limitations of each technology.

    • Application note

      Detecting insulin oligomerization using microfluidic diffusional sizing

      Insulin monomers self-assemble into hexamers, which is known to affect its level of uptake in the human body. Here we show that Microfluidic Diffusional Sizing (MDS) can be used to detect these changes.

    • Publication

      Latent analysis of unmodified biomolecules and their complexes in solution with attomole detection sensitivity

      In this paper, Yates et al show that the technology behind Fluidic Analytics instruments - microfludic diffusional sizing - can be used to accurately size a range of proteins.

    • Publication

      Microfluidic diffusion analysis of the sizes and interactions of proteins under native solution conditions.

      In this paper from Arosio et al microfluidic diffusional sizing (MDS) is used in combination with pre-labelled fluorescent biomolecules to size and quantify proteins in complex mixtures. Initially, MDS is compared to Dynamic Light Scatter (DLS) - and a good correlation is observed for monodispersed solutions.

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The Fluidity One

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

Fluidity One in use