Robustness to sample conditions:
There are basically no concentration or matrix constraints.
fida
Absolute molecular size
FIDA hydrodynamic radius measurement
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When understanding molecular behaviour is the goal, the hydrodynamic radius is the key. Why? Because it...
Captures the actual size and shape of molecules in solution, giving a clearer picture of how they behave in native conditions.
Reveals the true complexity of molecular structures, providing insights into how molecules fold, aggregate, and interact in solution
Reveals the true spatial dimensions of molecules in solution, allowing for a more accurate analysis of their dynamics and interactions in native conditions
Gives a complete functional picture of the molecule's size, conformation, and behavior in real-world conditions, making it a more versatile and informative measurement than molecular weight.
Absolute hydrodynamic radius provides direct access to information about:
In FIDA assays you can use any buffer, detergent, pH or ionic strength
The integrity and stability of the molecule and how it is impacted by changes in the environment ⓘ
Binding affinities, as well as the parameters that impact the affinity
Binding kinetics, as well as the parameters that impact the kinetics
How does Absolute Size refers to Non-Absolute Size measurements?
Highest accuracy:
The proprietary, highly-sensitive FIDA detectors ensure that also small changes are observed, even when working with polydisperse or complex samples.
No assumptions:
Absolute measurements are based on first principles, providing direct, quantifiable data without relying on calibration standards.
Technology
Precision
Sample Conditions
FIDA
High
Works with unpurified, complex samples (e.g. fermentation media, cell lysate, serum etc.); uses low volume (nanoliters to microliters); poses no buffer constraints on detergent, pH, ionic strength etc. - expanding the range of possible biological systems to be characterised. Furthermore, it quantifies aggregation and delivers direct (absolute), in-solution molecular size measurement.
DLS
Moderate
Requires purified samples, sensitive to dust and aggregates
SEC
Moderate to High
Sensitive to interactions with column materials, separates according to relative size - not absolute measure of size
AUC
High
Requires large samples amounts, and pure samples, can analyze in solution
Watch a video about FIDA
Size-based quality control: a wealth of information to accelerate your research
Size measurements are the fundament for over 10 parameters delivered by FIDA. Detection of hydrodynamic radius, changes in hydrodynamic radius and apparent hydrodynamic radius provide the measurements used not only for the Size, but also for Binding & Affinity, Binding Kinetics, Quantity, and Quality parameters, which you can read more about:
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FAQs
Would you like to know more? See frequently asked questions below. If you do not find an answer to your question, you can ask a question to one of our scientists.
How can you achieve the highest level of size measurement precision?
According to our specification, FIDA measures a size change of 5%. However, due to our proprietary detection system, there are many systems where FIDA reproducibly detects size changes of only 1%. This is possible thanks to two factors.
1. Firstly, FIDA’s Rh measurement is absolute, based on first principles, thus independent of assumptions and calibration. Read more about it here.
2. Secondly, the dispersion-based absolute size readout is supported by Binding Related Intensity Change (BRIC) measurement and Lambda Dynamics measurement (wavelength shift detection). Read about both readouts here.
How does hydrodynamic radius measurement differ from molecular weight?
Measuring the absolute hydrodynamic radius (Rh) of molecules offers several advantages over measuring molecular weight, particularly in understanding molecular behavior and interactions in solution. Hydrodynamic radius (Rh) and molecular weight are related, but they measure different aspects. While molecular weight tells you the mass of a molecule, Rh reflects its size in solution, accounting for shape, conformation, and hydration. Two molecules with the same molecular weight can have very different Rh values due to their structures (e.g., compact vs. elongated).
Here are some of the reasons why Rh is often of higher value or at least a complementary metric:
Direct measurement of size in solution:
Hydrodynamic radius reflects the actual size of the molecule in its native state, including any hydration shell and its overall conformation in solution. In contrast, molecular weight only tells you the mass of the molecule, which doesn’t account for its shape or how it behaves in a real biological environment.
For example, two proteins with the same molecular weight can have vastly different Rh values depending on their structural conformation (compact or extended).
Insight into Molecular Conformation
Rh provides direct insights into the 3D conformation of the molecule. This is particularly useful for studying proteins that can adopt different folded or unfolded states, as those states affect the hydrodynamic radius more than the molecular weight.
With molecular weight, you would miss information on whether a protein is compact, unfolded, or part of an aggregate.
More Relevant for Biological Systems
In biological processes, molecular interactions often depend on the physical size and shape of a molecule in solution rather than just its mass. Rh measurements are particularly relevant in studying processes like protein-protein interactions, aggregation, or binding events, which are dependent on the molecule’s actual spatial properties.
Aggregates and Complexes
Rh is highly useful for detecting and characterizing molecular aggregates or complexes. Aggregates often have the same molecular weight as their monomer counterparts but exhibit significantly larger hydrodynamic radii. This makes it easier to differentiate between monomers, oligomers, and aggregates in solution. Molecular weight alone might not reveal this, as it doesn't reflect the spatial arrangement or size of the complex.
Absolute Measurement
Hydrodynamic radius is measured as an absolute parameter, based on biophysical principles, and does not require assumptions or calibration against standards, as often needed in techniques used for molecular weight determination (e.g., gel filtration). This allows for more precise and reliable sizing of molecules in various environments.
Broader Range of Applications
Rh measurements are relevant across a wider range of scientific fields, from protein folding studies to aggregation analysis, drug binding assays, and quality control. They allow researchers to understand not just the size of the molecule but also how it behaves in dynamic systems, which is crucial in biophysical, pharmaceutical, and biotechnology research.
In summary, while molecular weight provides important information about the mass of a molecule, hydrodynamic radius gives a more complete and functional picture of the molecule's size, conformation, and behavior in real-world conditions, making it a more versatile and informative measurement.
Can hydrodynamic radius measurements be applied to study molecules beyond proteins?
Yes, hydrodynamic radius can be used to study a wide range of molecules beyond proteins, including nucleic acids, nanoparticles (LNPs), polymers, and even complex biological systems like viruses or liposomes. Interestingly, FIDA’s size measurement is even used for studying biomolecular condensates. It's a versatile tool for analyzing the size, structure, and interactions of various types of molecules in solution.
Can hydrodynamic radius measurements be used to study non-globular proteins, and how do they account for such structures?
Yes, hydrodynamic radius measurements are applicable to non-globular proteins as well. Unlike molecular weight, which only reflects mass, Rh provides insights into the actual conformation and extended structure of non-globular proteins. Since Rh measures how a molecule diffuses in solution, it accounts for elongated, flexible, or irregular shapes, offering a more accurate representation of the protein’s behavior in its native state.
Can FIDA’s hydrodynamic radius measurement be used to study how molecules respond to different environments?
Yes, Rh can be influenced by factors like pH, ionic strength, and the presence of detergents or other solutes. These factors may alter the molecule's conformation, hydration shell, or interactions, which in turn affect its hydrodynamic radius. This sensitivity can be leveraged to study how molecules respond to different environments.
Can I use hydrodynamic radius to study molecular aggregates?
Absolutely. Rh is ideal for studying molecular aggregates, as it can differentiate between monomers, oligomers, and larger aggregates. Since aggregates often have the same molecular weight as their individual components, Rh provides additional insight by revealing changes in size and conformation during aggregation. In FIDA aggregates show as signal spikes, meaning that you can also quantify them.
How is hydrodynamic radius complementary to molecular weight-based techniques like SDS-PAGE or mass spectrometry?
FIDA’s absolute in-solution Rh measurement offers high accuracy and gives a more functional insight into how the molecule behaves in solution. FIDA, for instance, measures Rh with precision and in real-time, offering a unique advantage over purely mass-based techniques. Moreover, due to buffer, detergent, ionic strength and pH flexibility, as well as the ability to study unpurified samples, it allows to simulate the native conditions of proteins.
Can I use FIDA’s absolute hydrodynamic radius to study small molecules?
Absolutely. According to our specification, FIDA measures a size change of 5%. However, due to our proprietary detection system, there are many systems where FIDA reproducibly detects size changes of only 1%. Thus, it is sucessfuly being used for small molecule research.
How does the hydrodynamic radius handle mixtures of molecules with varying molecular weights?
With FIDA, hydrodynamic radius measurements can effectively resolve and analyze mixtures of molecules with varying molecular weights. FIDA technology allows you to track specific components in complex mixtures by measuring the Rh of individual species, even when their molecular weights differ. This is particularly useful for monitoring interactions, binding events, or detecting aggregates, as FIDA can precisely differentiate between different molecular species based on their Rh, providing detailed insights into how each component behaves in solution
Learn more during an exploratory call with Fidabio team.
We are happy to answer all of your questions. Book an exploratory call to learn more about FIDA and the match between your personal needs and what we can deliver. The call is non-binding and free of any charges, so feel free to fill the form!
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When understanding molecular behaviour is the goal, the hydrodynamic radius is the key. Why? Because it...
Captures the actual size and shape of molecules in solution, giving a clearer picture of how they behave in native conditions.
Reveals the true complexity of molecular structures, providing insights into how molecules fold, aggregate, and interact in solution
Reveals the true spatial dimensions of molecules in solution, allowing for a more accurate analysis of their dynamics and interactions in native conditions
Gives a complete functional picture of the molecule's size, conformation, and behavior in real-world conditions, making it a more versatile and informative measurement than molecular weight.