Book a discovery call
Becoming and being a FIDA user
Protein analysis and characterisation
Protein stability and storage
Choose a category or type below.
Thank you! Your submission has been received!
Oops! Something went wrong while submitting the form.
Becoming and being a FIDA user
Protein analysis and characterisation
Protein stability and storage
How much bench space do I need? / What is the footprint?
Fida setup footprint with 1 detector: 120 cm x 70 cm Fida setup footprint with 2 detectors: 140 cm x 70 cm Note, that you will also need space for a PC.
What does FIDA Software include?
FIDA Software Suite includes an Assay design module, an Assay Setup Module (instrument controls), a Data Analysis Module and a Reporting module.
What Quality Control parameters are incorporated in the software?
8 QC parameters are directly incorporated to our software: - Size - Aggregation - Stickiness - PDI - PDB correlator - Sample loss - Viscosity - Labelling quality
How to work with sticky samples?
In FIDA it is immediately clear, when stickiness reaches a level which might disturb the data readout. Stickiness is not causing problems in FIDA. Moderate sticky material can be run with regular FIDA capillaries. With stronger stickiness, you may apply a dedicated, dynamic coating to regular capillaries or permanently coated capillaries. This can be purchased on shop.fidabio.com (registered users only)
There is water on the plate seal (due to the environment the instrument operates in). Is there a way of solving this?
Yes, there is a method for dehumidifying. Please contact email@example.com for a consultation
How to measure sample aggregation
Aggregation is usually measured as a function of a given parameter (time, different buffers, temperature etc.) all of which can be assayed with FIDA. The emergence of the different types of aggregates can then by quantified by measuring the number of spikes (insoluble aggregates) and the total fluorescent area of larger species (soluble aggregates).
What types of aggregates are there and how do they show in FIDA data?
Different types of aggregates can be observed with FIDA: soluble diffusive aggregates and insoluble non-diffusive aggregates. Soluble aggregates will register as a shoulder or bump on the Taylor-gram, while insoluble aggregates will register as a signal spike. The soluble kind can be quantified by their total fluorescent area by fitting multiple species. The insoluble ones can be counted by counting the spikes, as each spike is at least one aggregate, press to see the example.
Is labelling your molecule mandatory to perform a FIDA assay?
Why is my protein sample aggregating?
Reasons for protein sample aggregation can vary. Press to see the answer.
How does Fida 1 quantify aggregation?
FIDA measurements automatically provide quantification of aggregation through a spike count (see image below).
What is protein aggregation?
Aggregation in molecular biology refers to the degradation process by which biomolecules, such as proteins or nucleic acids, cluster together, often forming larger complexes. After synthesis, proteins typically fold into a particular three-dimensional conformation (native state). This folding process can be impaired, creating protein misfolding or unfolding and lead to the protein aggregate. Keep reading...
Is the Fida 1 or FIDA software Suite compliant with 21 CFR part 11?
A dedicated 21 CFR part 11 FIDA Software Suite is available. Contact your regional sales representative for more information.
Is there a licence on Fida 1 or FIDA software Suite?
Currently, the FIDA software is not separately charged.
How easy is it to perform an experiment with FIDA? Do you need to be an expert?
Both hardware and software are user-oriented and easy to use. After a day of training, you are autonomous on the Fida 1. Our team provides regular training and advice, allowing our users to upgrade their skills and have new users trained regularly.
What are the QC parameters directly incorporated into the software?
There are 8 QC parameters that are directly incorporated to our software: - Size - Aggregation - Stickiness - PDI - PDB correlator - Sample loss - Viscosity - Labelling quality
What are the assay design tools?
Assay design enables in-silico assay development and protocol generation. All the relevant parameters can be documented, processed, and evaluated upfront ahead of wet lab experiments. The assay design tool helps the user to find the methods specifically for its sample for optimal data generation. For ease of use, users can generate protocols in .doc format containing all defined information as well as detailed dilution series for sample preparation. Assay specific methods and sequences can be exported directly to the Fida 1 instrument, which saves time and minimises risks of human error.
How are the fitting models calculated?
Different binding models are used in the binding curve module in the Fida data analysis software. In general, the binding models are derived assuming that the apparent diffusivity of the indicator is a weighted average of the fractions bound and unbound of the indicator. The exported data can then be treated in generic spreadsheet-based analysis software and custom graphics can be designed for presentations and publications. Press to read more and see calculations.
Is there a mandatory maintenance on Fida 1?
Fida 1 is a very robust system which does not require calibration or day-to-day maintenance in addition to standard laboratory tidiness or cleaning. To ensure consistently high performance, we do provide service agreements with preventive maintenance etc. Ask your regional representative for an offer. If you need any technical help contact firstname.lastname@example.org
What is the temperature range on Fida 1?
There are three independent temperature control: The temperature of the capillary compartment, as well as the two sample trays. The sample trays can be independently set from 5 up to 55 degrees +/- 1 degree. The capillary compartment can be set between 10° Celsius - 55° Celsius. Cooling to 5° Celsius assumes a room temperature at 22° Celsius or below.
Are the 96-well plates from FIDA compatible with a pipetting robot?
Yes, the 96-well plates from FIDA are compatible with pipetting robots
How much time is typically needed to get a KD using the Fida 1?
It takes from app. 30 min. depending on the assay type.
What types of coating are there?
We offer have 2 types of coating: -Dynamic coating (can be applied and removed on an uncoated capillary) - Permanently coated capillary The dynamic coating presents a hydrophilic, non-charged surface and can be stripped and reapplied. Therefore, even chemicals that would strip the coating can be used with this type of a capillary. There are no restrictions on pH with this coating. The permanent coating is making the surface of the capillary more hydrophilic and shields the charges, showing higher performance in reducing stickiness at neutral pH. However, pH > 10 destroys it and it becomes protonated at pH <5.Purchase at shop.fidabio.com (registered users only)
What is the size of the capillary?
Capillaries have 75µM of diameter and 1m length, on special request of the customer, we can provide capillaries of other length or diameter
What type of capillaries are there?
Uncoated and permanently coated.
What are the sample formats available on Fida 1?
There are two formats available both handled by an autosampler: 50 x 1.5 ml glass vials where you can add an insert for small volume or two 96 well plates.
What is the volume consumed by Fida 1 per sample?
Naturally, the consumption varies by application. In general, it consumes down to 4µl of analyte and 40nl of indicator per measurement.
Is there any upper limit in terms of salt concentration?
No, there is none.
Does viscosity impact the final Fida 1 read-out?
No, FIDA provides precise viscosity measurements and any change in viscosity will be accommodated for in the FIDA data analysis. The Fida 1 can also be used as an advanced, highly accurate viscosity meter.
Is it possible to work in PEG (viscous liquid) Glycerol, DMSO media with FIDA?
Yes it is. As the viscosity is measured and compensate at every measurement, high percentage of viscous additive (glycerol, DMSO, PEG…) can be added to your buffer prior to a FIDA measurement.
Is there any Ph buffer restriction to use Fida 1?
No. Contrary to many other technologies, FIDA provides full buffer flexibility, incl. PEG, Glycerol, DMSO media etc. As the viscosity is measured and compensated at every measurement, a high percentage of viscous additives can be added to your buffer prior to a FIDA measurement.
Is there any buffer restriction on Fida 1?
No, there is no buffer restriction with FIDA. Hurra! Crude matrix like serum, plasma, fermentation media, cell lysate works perfectly fine on FIDA, which increases clinical relevance of the studies made. On top of that, as the viscosity is measured and compensate at every measurement, high percentage of viscous additive (glycerol, DMSO, PEG…) can be added to your buffer prior a FIDA measurement.
How to measure binding affinity?
Measuring binding affinity is achieved through Flow Induced Dispersion Analysis (FIDA) by quantifying changes in the hydrodynamic radius (Rh). As biomolecules bind in a solution, their diffusivity decreases, leading to slower diffusion and an extended dispersion profile. FIDA software, applying the Stokes-Einstein equation, precisely determines the increase in size (Rh) as complexes start to form. By titrating the molecule of interest with its binding partners in a straightforward FIDA experiment, binding affinity (Kd) can be accurately assessed based solely on the hydrodynamic radius and the Stokes-Einstein equation.
What is the relationship between Rh and molecular weight?
Molecular size and molecular weight are the two most used properties to characterise molecules. Molecular size can be approximated with molecular weight. Molecular size refers to the geometry of the molecule and is reported as the hydrodynamic radius (Rh). Rh is the absolute size, in nanometers, of the molecule in solution. On the other hand, molecular weight is calculated as the sum of the atomic weights of all the atoms in a molecule and is reported in kilodaltons (kDa).In general, larger sized molecules tend to have higher molecular weight than smaller molecules, but the relationship between molecular size and weight is not always straightforward. This is especially true when considering different conformational states of proteins, such as globular versus unfolded, as the complexity of measurements increases in this case. Press to keep reading...
How precise if FIDA’s hydrodynamic radius (Rh)?
FIDA can detect size changes smaller than 5%. In many cases you may detect changes down to an angstrom.
What information can hydrodynamic radius provide?
Press to load full answer.
What is hydrodynamic radius?
Hydrodynamic radius (Rh), is a parameter used to describe the size of a particle or molecule in a fluid, taking into account its shape, mass, and interactions with the surrounding fluid molecules. The hydrodynamic radius is defined as the radius of a hypothetical sphere that diffuses at the same rate as the particle or molecule in question, under the same conditions (Einstein, 1905).
What are the two main FIDA read-outs?
The 2 main read-outs with FIDA are: - The Hydrodynamic radius (size in nm) - The Relative Fluorescence Unit (RFU)
How does Fida 1 detect a signal?
FIDA requires a fluorescent signal coming from your sample that can be detected by one of our detectors. It can either be emitting from a label or from the molecule itself, i.e. label-free. The signal coming from your sample that can be detected by one of our detectors: 480, 550, 640, 280. FIDA uses a LED excitation and amplifies the emission signal by PMT. Therefore, FIDA does not use any laser to get a signal, and the sample is not heated. Note that FIDA delivers two orthogonal readouts: absolute size (hydrodynamic radius) and fluorescence.
Why analyse in-solution?
In-solution studies increase data reliability, due to their ability to mimic the natural physiological conditions in which proteins and particles function. Proteins and particles are sensitive structures. Their functions are often impacted by environmental changes, by labelling or by immobilization to a surface structure and functionality of the protein. All-in-all, in-solution studies increase data reliability, due to their ability to mimic the natural physiological conditions in which proteins and particles function. In solution approach allows to better understand the dynamic molecular interactions, conformational changes, and biochemical reactions, leading to more accurate insights into their behavior and enabling the development of more targeted and effective applications in various fields, including drug discovery, diagnostics, and biotechnology.
What are the first principles that FIDA is based on?
FIDA is based on 2 principles: Taylor Dispersion Analysis & Stokes-Einstein Equation. Utilising Taylor Dispersion Analysis, incl. the Stokes-Einstein Equation, FIDA in a very accurate way detects the change in apparent diffusivity that occurs when an analyte interacts with a binding partner or the environment changes, which causes a change in the hydrodynamic radius readout. Press to keep reading.
What is FIDA?
FIDA stands for Flow Induced Dispersion Analysis. It is a first-principle based, in-solution technology, which provides a precise, quantitative, and highly robust method to determine how changes to an analyte affect the analyte. The changes that may be assessed are numerous ranging from adding a binding partner to changes in the environments including ionic strength, pH, buffers and different crude matrixes, temperature etc. The typical readout is hydrodynamic radi which provides information about quantity, affinity, sample integrity, changes in oligomeric state, aggregation, stoichiometry etc. Press to keep reading...
How to perform a binding affinity assay?
FIDA is a fluorescence-based technique, hence for a binding affinity assay we need a protein with intrinsic fluorescence or a fluorophore on it. This is what we call the indicator and allows us to observe the interaction between an indicator and a binding partner. The binding partner is what we call the analyte. During a binding assay, the indicator is kept at a constant concentration and the analyte is titrated in at increasing concentrations. The analyte concentration should be high enough to reach a plateau on a binding curve to ensure full complex formation. This would allow you to extract the size of the indicator alone, the size of the indicator-analyte complex, and the binding affinity between indicator and analyte.
How to study protein cooperativity?
Using the FIDA technology you will be able to study ternary complex formation, where the cooperativity is one of the parameters extracted. On top of the cooperativity you will also gain information about the size of the complex and the affinity of the interactions.
How to measure protein regulation?
If a significant size change is caused by the regulations, it can be measured using FIDA. Alternatively, if the regulation is intended to increase affinity toward a target, FIDA can also be used to quantify whether the regulation increased the affinity or not.
How to measure protein unfolding?
FIDA can measure the unfolding of a protein through chemical denaturation. As we have no limitation on the matrix you measure your sample in we can use various chemical denaturants, such as urea or GnCl, to unfold the proteins.
How to optimise protein storage?
You can use the Fida 1 instrument to measure the stability of your protein. This means that you can store your protein in different ways and after storing for some time, check the stability of the protein. The storage method giving the same/similar result as when the protein was freshly prepared will be the most optimal storage condition.
Can FIDA be used to measure thermal stability of proteins?
To an extent, yes. FIDA can control the temperature in the Fida Instrument in three different compartments: sample tray, buffer tray and the capillary chamber. In the sample and buffer tray the temperature can be controlled between 5 and 55 degrees Celsius. In the capillary chamber the temperature can be controlled between 10 and 50 degrees Celsius.
How to measure protein stability with Fida 1?
By using our UV-fluorescence detector you can measure the stability of your protein in any buffer system using only 40 nL in approximately 5 minutes. You simply load the protein into the Fida 1 instrument, run your experiment and the readout will tell you the size of your protein and, e.g., if there are any aggregates. If the protein structure is known remember to use the PDB correlator to extract information on the oligomeric state of the protein. Read application note for more.
What kind of optimization can be tested to minimize stickiness?
Fidabio data analysis software can tolerate mild stickiness without impacting the readout. In cases of strong stickiness between the sample and the capillary there are a few methods that can be used. It’s important to note that the capillary wall is made of fused silica, hence it will have an induced negative charge. This can cause interactions with proteins. To overcome this, you can: 1. Coat the capillary with our dynamic coating solution 2. Use our permanently coated capillary. 3. Modify the buffer with e.g. salt or change the pH to limit the interaction between the proteins and the capillary wall.
What do when sample is sticky?
Fidabio data analysis software can tolerate mild stickiness without impacting the readout. In cases of strong stickiness between the sample and the capillary, a permamently coated capillary or a reversible surface coating can be can be used. For more on stickiness read the article For capillary or coating purchases visit shop.fidabio.com (registered users only)
Do sticky proteins impact the FIDA signal?
When a protein is sticking to the capillary walls it will change the readout. We typically observe stickiness on the readout as a skewed gaussian signal. The software will indicate the stickiness issue, so you will be able to solve it.
Can FIDA identify stickiness and issues related to it?
Yes, Fida Software can indicate if the sample is sticking to the capillary walls. it can also indicate whether the issue os after assay optimisation.
What different types of protein analysis/assays can be done with FIDA technology?
Press to see the full list
How many runs (measurement) can be done per capillary?
We recommend running up to 400 runs per capillary. Overusing this consumable might impact the results.
After I become a user, where will be able to purchase consumables?
The consumables can be purchased on shop.fidabio.com (webshop accessable after you become a user)
How does the FIDA user community work?
Our users can participate in Advanced User training, organised bi-monthly in Copenhagen, Denmark and every few months in Cambridge, Boston, USA. On top of that all the users are invited to participate in global and local User Group Meetings, where they can receive extra training, workshops, and knowledge sharing sessions, as well and networking and entertainment. Our users come from diverse industries, which stimulates knowledge dissemination and innovation potential. Note that the Advanced User Training Course is for free, however we do not cover the travel and accommodation costs.
Is the Fida 1 analysis software suite freely available to FIDA users?
Yes, including ongoing updates.
How to become a FIDA user?
The process is simple and straightforward, and our designated FIDA specialist will be with you throughout the whole process. Press for more.
What is stickiness?
Stickiness is an unspecific interaction between a molecule and e.g. a surface. The stickiness of biomolecules is influenced by various factors, including the presence of specific binding sites, electrostatic interactions, van der Waals forces, and hydrophobic interactions. Knowing the stickiness of your sample, enables you to ensure that you are actually measuring the molecule or the complex, you want to analyse and avoid misinterpretations of your data. Read more here.
Why use PDB Correlator as a QC tool?
After an experiment on a Fida 1, you can use the PDB correlator as an orthogonal method of validating the state of your protein. If the Fida 1 results and the PDB correlator agrees, you then you have the oligomeric state of your protein confirmed. If it differs you know that your protein in solution behave differently compared to the crystal structure. Read more here.
How to correlate Alphafold data?
By using Alphafold you can generate a PDB file based on the protein sequence. This allows you to use our PDB correlator - read more here.
How does the PDB Correlator work?
The PDB correlator extracts the atomic coordinates from a PDB-file and and based on our algorithms it predicts the hydrodynamic radius, radius of gyration, shape of the protein, number of atoms and molecular weight. It can be used as a quality control tool e.g. to confirm that the sample is indeed what is expected, to determine oligomeric state, etc. Keep reading...
Can I assess different binding stochiometry with Fida 1?
Yes. Different fitting models are available in Fida Software. These can be applied to the obtained binding curve based on if the interaction is 1:1, 1:2 or 1: 3 type.
Is it needed to centrifugate your sample prior to FIDA measurement?
No, in general, it is not mandatory to centrifuge purified samples prior to FIDA measurements. However, if data retrieved by FIDA runs indicates presence of large aggregates (seen as signal spikes), samples can be centrifuged before performing binding assays. Moreover, centrifugation of sample might be necessary when working with crude cell lysates, to get rid of large fragments or cell debris. FIDA Quality Control, such as quantified aggregation measurement can be useful to determine this need.
Can I do triplicate measurement from the same sample preparation?
Yes, technical replicates from the same sample preparation can be performed by assigning the desired replicate count on the software interface. The only requirement here will be the availability of sufficient analyte solution for replicate runs. It is advised to load at least 40 ul of analyte for triplicate runs.
Can the sample be reused?
Yes! Thanks to thermally regulated sample loading chamber on a Fida 1 loaded set of samples can be incubated at desired temperature (e.g. 4°C) for entire duration of measurement. Post-measurement, the remaining sample volume can be retrieved back from the loading chamber. This set up allows for sample savings.
Can Fida 1 test serum samples without the measurement being negatively impacted by the background noise?
Analyte solution used in a Fida 1 measurement is recorded as base line signal and indicator as the gaussian peak. In cases where serum samples are used as analyte, the background noise gives higher baseline signal, still enabling the analysis of indicator peak. Press to see the image.
How does FIDA handle non-globular proteins?
FIDA, or Flow Induced Dispersion Analysis, is a technology that addresses the measurement of non-globular proteins without making any structural assumptions about the molecules. This unique approach sets FIDA apart, as it doesn't rely on a priori knowledge of the molecular structure. Instead, FIDA measures the hydrodynamic radius (Rh) of species, which can be both globular or non-globular, including intrinsically disordered or partially unfolded proteins. Keep reading...
What is the biggest molecule that can be measured with Fida 1?
The largest species that can be measured with Fida 1 can be ~500 nm in Rh or 1 um in Dh. These include species like exosomes, liposomes, VLPs, AAVs, etc.
What are the size measurement limits?
The smallest molecule that can be measured by Fida 1 can be down to 0.5 nm in hydrodynamic radius (Rh). This could be a species like small biomolecules or peptides. The largest molecules that can be measured by Fida 1 can be species of up to 500 nm in Rh. This includes large nanoparticles (like LNPs, OMVs, etc.), liposomes, exosomes or virus-like particles.
Can Fida 1 characterise small molecule binding?
Yes: FIDA measurements delivers 2 major orthogonal readouts, I.e., the absolute in-solution hydrodynamic radius (Rh) and fluorescence intensity. In the case of small molecule interactions, FIDA assays can be performed in 3 different ways where: 1. Global conformational changes upon small molecule treatment can be detected as a change in Rh. 2. Small conformational changes that do not necessarily lead to change in global size of the target, could be detected as change in Tryptophan fluorescence at 280 nm excitation. 3. Small molecule interactions can be analysed as shift in denaturation point by performing chemical unfolding assays in presence and absence of small molecule on Fida 1. Read application note: ‘Exploring Small Molecule Interactions with Membrane Proteins’
What is the sample usage?
Sample consumption can be different for different FIDA assay setups. 1. For standard binding assays – 40 nL of low nM range indicator and 12 ul of analyte is consumed per run. The concentration of analyte needed will depend on the affinity of interaction. 2. For sample Quality Control – 40 nL of uM range indicator and 12 ul of the buffer analyte is consumed per run.
Does viscosity impact FIDA measurement?
Sample viscosity is measured with every single run and is automatically compensated for Rh measurements. Plus, Fida 1 autosampler enables accurate temperature control in the measurement chamber and hence offers precise viscosity control. Thus, the impact of viscosity on the measurement is prevented.
Is it possible to work in viscous media (PEG, Glycerol or DMSO) with FIDA?
Fida 1 gives complete flexibility with the use of buffer additives. Additives which may skew sample viscosity can be used without any pre-calibration.
Why measure sample viscosity?
In most biophysical methods, a change in viscosity will impact the readout. Thus, to get correct readouts, you have to identify if there is a change in viscosity and compensate for the change. With hydrodynamic radius (Rh), for instance, an increase of viscosity can lead to over-estimation of Rh and vice versa. The Fida 1 automatically detects viscosity changes and compensates for such. Fida 1 measures sample viscosity with every run and enables automatic viscosity correction of Rh, without any requirement of manual post-processing of the data. Keep reading...
No question or topic found.
Send us an email
In case you need
Call us now
(+45) 53 72 78 70