Quality Control

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...

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...

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.

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.

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.

In a Fida 1 experiment a sticky sample is when it is interacting with the fused silica that is the capillary wall.

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.

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.

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)

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.

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.

There are 8 QC parameters that are directly incorporated to our software: - Size - Aggregation - Stickiness - PDI - PDB correlator - Sample loss - Viscosity - Labelling quality

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...

Reasons for protein sample aggregation can vary. Press to see the answer.

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.

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).

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)

8 QC parameters are directly incorporated to our software: - Size - Aggregation - Stickiness - PDI - PDB correlator - Sample loss - Viscosity - Labelling quality

Autosampler temperature control: 5-55°C (41°C-131°F) Capillary chamber temperature control 15°-55°C (59°-131°F)

You can export your QC data as .txt or PDFs

An unconjugated label is a much smaller size than a label conjugated to a biomolecule. In FIDA one can identify multiple species and their relative abundance. This is the feature used to give labeling quality.

No, over-labeling will have no impact on the sizing measurement using Fida. However, mind that for all technologies that rely on labelling to detect interactions over-labelling risks putting a label in or nearby the binding site inhibiting the binding due to stearic hindrance.

Yes, there are several non-covalent labels that will work examples that has been applied in the past are: - HIS Lite™ OG488-Tris NTA-Ni Complex against the His-tag. - Single domain antibodies against the ALFA-tag. - As well as nanobodies specific for your protein of interest.

Polydispersity Index (PDI) is a crucial parameter that describes the relative distribution of molecular sizes in a sample. Unlike substances with uniform molecular size, some materials, such as aggregates and biopolymers, exhibit a range of lengths and sizes. The PDI quantifies this variability, providing insight into the sample's heterogeneity.A PDI value close to 1 indicates a uniform sample, where all molecules are nearly the same size. Higher PDI values signify a broader distribution of molecular sizes, indicating greater variability within the sample. Understanding PDI is essential in fields like polymer science and biotechnology, where the size distribution of molecules can impact material properties and functionality.

Protein oligomerization refers to the process by which two or more protein molecules (monomers) associate to form a larger, often functional complex called an oligomer. These oligomers can be composed of identical (homooligomers) or different (heterooligomers) protein subunits. This process is critical in various biological functions and is fundamental to the structure and activity of many proteins.