Measuring Up: How Hydrodynamic Radius is Revolutionising Biophysical Research
Imagine there being one parameter that could provide valuable insights into numerous biomolecular properties, such as affinity, titer, oligomerization, conformational changes, and immunogenicity. As evidenced in the recent years, hydrodynamic radius is becoming a key parameter, above and beyond “just” sizing.
In this article from FIDA Quality Control Series, we will highlight how sizing is used in biophysical analysis.
What is hydrodynamic radius (Rh)?
Hydrodynamic radius (Rh) (also called the Stokes radius) is a measure of the size of a molecule in solution determined by means of its diffusion coefficient. With modern technologies, it is not limited to spheres. The diffusion coefficient can be determined by a number of relative and absolute technologies that can work with purified and complex matrices and with labelled as well as unlabeled samples. For accurate radii determination, it is essential to have a precise measurement of the diffusion, can exclude aggregates and can compensate for viscosity. Precise measurements for Rh can provide essential information about simple molecules and complex constructs, incl. affinity, quantity, oligomerization, agglutination, ternary complex formation, conformational changes, buffer screening, clone selection, immunogenicity, and it is a key QC parameter.
Hydrodynamic radius (Rh) (also called the Stokes radius) is a measure of the size of a molecule in solution determined by means of its diffusion coefficient.
Why is size a key QC parameter?
However, in biophysical studies also size, in itself, is a unique quantitative source of information about the overall conformation and structure of proteins and their complexes.
Especially when using truly quantitative technologies, the size is directly linked to the structural integrity of the analysed sample and, thereby, to information about degradation, stability, oligomerization, agglutination, conformational changes etc. In other words, if a researcher knows the precise size of the molecule they are analysing, f. ex. in a binding study, they have a strong indication of the reliability of the affinity data they are getting, and can avoid assumptions.
Why study size in solution?
Proteins and particles are sensitive structures. Their functions are often impacted by environmental changes, by labelling or by immobilization to a surface. Sizing technologies allow researchers to study their candidates in solution and, in many cases, under native or near-native conditions. Thereby they preserve the molecular structure and functionality of the protein. All-in-all, in-solution studies increase data reliability.
How does FIDA measure the hydrodynamic radius?
FIDA technology applies first principle thinking. It uses transparent and verifiable physics and fluid mechanics to determine the diffusion coefficient and the size. Thereby, it is easy to optimize assays and validate data. With FIDA, researchers can work with labelled as well as unlabeled samples.
See our video explainer to understand the technology in 5min!
What is FIDA's sensitivity?
FIDA allows its users to work within a size range of 0,5-500 Rh. The technology detects size changes of less than 5%, and can often reproducibly detect changes in the 1% range. I.e., it enables the user to see subtle folding/unfolding and other conformational changes. Automated viscosity determination and compensation, as well as clear identification of potential aggregates, further increases sensitivity and data reliability.
How is size used as a quality control parameter in FIDA?
FIDA automatically generates all QC parameters with every analysis, and they are displayed in the Fida 1 Software Suite user interface. I.e., the user will immediately see if the size is in line with their expectations. As an extra feature, the Fida 1 Software Suite has a built-in PDB correlator which enables users to refer their size measure with the Protein Data Base structure. It is also possible to import proprietary structures or structures generated using Pymol or AlphaFold.
How much sample and what type of sample do I need to measure Rh using FIDA?
A few nanoliter amounts of sample is enough for a FIDA measurement. It is fully temperature controlled, and there are no pH or ionic constraints. It also works with complex matrices such as cell lysate, fermentation media or plasma. FIDA can deliver a Rh readout even in case of sticky proteins or viscous samples - more on that in our upcoming Quality Control articles; follow us to stay updated.
Would you like to calculate your hydrodynamic radius from molecular weight or your molecular weight from hydrodynamic radius (size), try our calculator: https://www.fidabio.com/molecular-weight-to-size-protein-radius-calculator
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