Sample

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.‍

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’

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.

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.

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.

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.

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.‍

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.

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.

Naturally, the consumption varies by application. In general, it consumes down to 4µl of analyte and 40nl of indicator per measurement.