Assay optimization


Adsorption

Biological molecules, especially proteins can easily attach to plastic labware by adsorption.

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Aggregation

Aggregation describes the formation of large clusters of biomolecules. Dianthus software helps to identify aggregates and particles since they are responsible for irregular TRIC-trace.

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Buffer

In general, there are no limitations for the assay buffer used in TRIC experiments. Typically, in vitro biochemical and biophysical assays are performed at near physiological pH in an attempt to mimic the native environment of the protein.

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Buffer Autofluorescence

Most commonly used biological buffers (see buffer) don’t show a significant fluorescence signal at the red emission wavelengths, relevant for Dianthus experiments.

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Buffer Gradients

Serial dilution of the ligand in assay buffer should not introduce any changes in buffer composition except the ligand concentration itself.

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Competition Assay

A competitive binding assay typically measures the binding of a labeled ligand to a target protein in the presence of a second, competing but unlabeled ligand.

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Concentrations

Target: To achieve a good resolution of the binding event, the concentration of the fluorescent target needs to be lower or in the range of the Kd of the interaction.

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Control Experiment

Control experiments for TRIC ideally use a system that is very similar to the one under investigation.

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Detergents

Some detergents can be used to solubilize recombinant proteins, while others are recommended for the stabilization, crystallization, or denaturation of proteins.

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DMSO

DMSO Dimethyl sulfoxide (DMSO) and Dimethylformamide (DMF) are the preferred solvents for organic small molecules.

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Fluorescent fusion proteins

A protein can be fluorescently tagged by expressing it as a fusion with a fluorescent protein (FP). Only a few fluorescent proteins are available with far red emission spectra.

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Fluorophores

A fluorophore is a molecule with fluorescence properties. The fluorophore absorbs photons and emits photons of lower energy in return.

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Freeze Thaw Cycles

Repeated freezing and thawing of biomolecules such as proteins can lead to significant denaturation even in the presence of cryoprotectants, therefore aliquotation of protein preparations is generally recommended.

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Ligand Autofluorescence

TRIC uses changes in the temperature dependence of a measured fluorescence signal to qualitatively and quantitatively analyze molecular interactions.

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Ligand-Induced Fluorescence Change

TRIC detects biomolecular interactions by following changes in target fluorescence upon binding of ligands. Ligand binding causes a change in the TRIC signal of the target, which enables binding detection.

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Ligand-Induced Photobleaching

In some rare cases, ligand binding can change the photobleaching properties of the fluorescent target. This means that the photobleaching behavior changes with the ligand concentration (see concentrations).

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Liquid Handling

The accuracy of liquid handling systems can greatly impact the quality of the resulting data.

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PEG 8000

Polyethylen glycols (PEGs) are amphiphilic polymers composed of repeating ethylene glycol units. Used as additives, they can improve sample quality by preventing surface adsorption and aggregation of biomolecules.

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Positive Control Sample

In the context of single-dose screening and affinity screening, a positive control sample corresponds to a ligand sample that is a well-known or well-characterized interactor of the target (sometimes also called tool compound).

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Protein concentration and DOL

After protein labeling with a fluorescent dye (see fluorophores ), it is strongly recommended to determine the protein concentration and labeling efficiency.

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Protein Labeling

One of the most common ways to make a given target protein fluorescent is to attach organic fluorophores by means of different labeling chemistries.

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Quenching

Quenching refers to any process, which decreases the fluorescence intensity of fluorophores.

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Random Changes in Initial Fluorescence

Random changes in initial fluorescence are usually the result of sample inhomogeneities (such as target aggregation). Dianthus software recognizes random fluorescence changes and highlights them.

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RED-tris-NTA

The hexahistidine-tag (His6, see His-tag) provides binding sites for three Ni-NTA moieties. Tris-NTA is thus perfectly suited for non-covalent, stable, highly selective protein labeling of hexahistidine tags in a stoichiometric ratio of 1:1 at sub-nanomolar affinity.

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Reducing Agents

The addition of a reducing agent to an assay may be important to prevent the oxidation of cysteines in the investigated proteins and thereby the formation of disulfides. The most commonly used reducing agents are dithiothreitol (DTT), β-mercaptoethanol (β-ME) and tris(2-carboxyethyl)phosphine (TCEP).

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Reference Sample

In the context of a single-dose screening, a reference sample corresponds to the target in the unbound (see baseline) state.

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Serial Dilution

A stepwise dilution of ligand in buffer. Typically, a serial 1:1 dilution (dilution factor 2 in DI.Control software) is performed by transferring one volume of ligand solution to an equal volume of buffer, mixing, and repeating this step.

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Data interpretation


Aggregation Quality

Two different algorithms are used to detect aggregation from a TRIC-trace. In a binding check experiment or a single-dose screening the fluctuations are quantified, occurring within the TRIC trace.

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Area Response

The area response represents a robust approach to analyze the response amplitude and the signal quality of a TRIC assay.

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Baseline

The part of a TRIC dose response curve where the target is in the unbound state is also called baseline.

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Binder

Binder is a ligand category in DI.Screening Analysis software. It is a ligand that showed an interaction to a target molecule in an affinity screen.

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Dose Response Curve

For binding affinity analysis, ligand-dependent changes in TRIC are plotted as Fnorm values vs. ligand concentration (see concentrations).

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EC50

The parameter EC50 abbreviates for ‘half maximal effective concentration’. In a pharmacological context, this can be the concentration of a drug that is necessary to cause half of the maximum possible effect.

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ECP-Test

If a ligand-induced fluorescence change that exceeds ±20% is observed with NanoTemper Technologies RED-tris-NTA fluorophores, performing an ECP-test is advisable. The ECP-test is a type of specificity test.

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Fnorm

For binding affinity analysis, ligand-dependent changes in TRIC are plotted as Fnorm values vs. ligand concentration in a dose-response curve. Fnorm values are plotted as parts per thousand (‰).

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Fluorescence Quality

The NanoTemper Technologies Software products DI.Control and DI.Screening Analysis are both able to automatically detect issues with assay quality, such as initial fluorescence and use quality checks.

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Hill Model

The Hill fit is used for affinity quantification of multivalent interactions and provides information about the degree of cooperativity.

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Hit

Hit is a ligand category.

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Inconclusive

In the DI.Screening Analysis software ligands that were measured in TRIC screenings are automatically categorized to select for hits / binders and to single out non-binders or ligands that cause artifacts, such as aggregation of the target molecule.

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Inhomogeneity Quality

In single-dose screening all data points for a ligand have to show a consistent significant deviation from the references for the ligand to be considered a hit.

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Initial Fluorescence

In TRIC experiments, the initial fluorescence denotes the fluorescence of a sample that is measured before the IR- Laser is switched on, i.e. at ambient temperature without additional sample heating.

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Kd

The Kd is used as a measure of the affinity of an interaction.

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Kd Fit Model

The Kd fit model describes a molecular interaction with a 1:1 stoichiometry according to the law of mass action.

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Ligand Category

In DI.Screening Analysis software ligands are automatically assigned a category, depending on the data obtained for a particular ligand. The two main categories are hits and non-binders that are assigned based on the response amplitude measured.

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Merge Sets

Version 1.1 of DI.Screening Analysis software can work with merge sets of binding affinity data from affinity screening. A merge set is the merged dataset of replicates of a serial dilution.

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Non-Binder

In single-dose screening a ligand is considered a non-binder if it yields results in TRIC measurements that are similar within a certain user-defined threshold to the reference sample.

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Not Reproducible

“Not reproducible” is a ligand category in DI.Screening Analysis software that is assigned to a ligand in single-dose screening if replicates of the ligand show results that are not in agreement with one another.

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Outlier

In some cases DI.Screening Analysis software can single out problematic wells in affinity measurements (e.g. an outlier in initial fluorescence, in photobleaching or also Fnorm) and exclude them from analysis (not from view in charts).

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Photobleaching Quality

The NanoTemper Technologies Software products DI.Control and DI.Screening Analysis are both able to automatically detect issues with assay quality such as ligand-induced photobleaching and use quality checks.

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Potential Hit

Potential hit is a ligand category in single-dose screening and affinity screening. It is a sub-category of the inconclusive ligand category.

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Response Amplitude

Difference between TRIC signal of target and complex in ‰ Fnorm (affinity measurements) or area response (single-dose measurements).

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Response Quality

The final step in assessing a ligand dose response curve in DI.Screening Analysis software is to evaluate the response amplitude, resulting in the response quality check (see quality checks).

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Saturation

The part of a TRIC dose response curve, where the target is in the bound state is also called saturation.

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SD-Test

The SDS denaturation test (SD-Test) is a specificity test that was developed for the analysis of the source of a ligand-induced fluorescence change that exceeds ±20% of the fluorescence average.

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Signal Quality

The signal quality is a measure of assay quality and is applied to area response charts in binding check and buffer screen experiments.

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Signal-to-noise ratio

The signal-to-noise ratio is used to evaluate the quality of Fnorm data. It is defined as the response amplitude divided by the noise of the measurement.

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Specificity of an interaction

In rare cases, increasing concentrations of a ligand can cause changes in TRIC signals that are not specific (not binding-induced).

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Specificity Test

When a ligand-induced fluorescence change is detected that exceeds ±20%, it is recommended to perform a specificity test to distinguish between specific and non-specific effects.

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TRIC-trace

A TRIC-trace shows the change in fluorescence intensity of one well over time, while a temperature increase is applied by the IR-Laser.

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Weak Binder

Weak binder is a ligand category in DI.Screening Analysis software that is specific to affinity screening.

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Well-Scan Quality

The NanoTemper Technologies software products DI.Control and DI.Screening Analysis are both able to automatically detect anomalies of well scans for X- and Y-scans and display detected anomalies to the user.

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Dianthus software and system


Affinity Screening

In affinity screenings (sometimes also secondary screenings), in contrast to single-dose screening, the major goal is not to identify interactors but rather to determine the affinity for known binders.

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Binding Affinity

The binding affinity templates in DI.Control software can be used to quantify any molecular interaction between a fluorescently labeled target molecule and a corresponding ligand molecule.

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Binding Check

The binding check templates in the DI.Control software represent the fastest and easiest way to iteratively test and improve assay quality of a TRIC binding assay.

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Buffer Screen

The buffer screen experiment template in DI.Control relies on the use of the NanoTemper Technologies Buffer Exploration Kit (Cat# NT-B001).

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Data Export

Dianthus software solutions allow users to export all measured raw data, fitted curves, graphs and plots as well as metadata such as the result of quality checks.

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Dilutionseries ID

The dilution series ID signifies all wells that are considered part of a dilution series.

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Excitation Power

The excitation power is the strength of the LED used for excitation of fluorophores. In DI.Control the excitation power can be set to auto-excitation or to a manual setting.

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Exclude

The exclude function in DI.Screening Analysis can be used to exclude wells/data points from analysis.

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Filterset

Dianthus instruments are available with a red filterset that can be combined with a multitude of commercially available fluorophores.

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Import

The import experiment templates for affinity screening and single-dose screening allow to import plate layouts directly from Microsoft Excel.

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IR-Laser

In TRIC assays an infra-red (IR) laser with a wavelength of 1480 nm is absorbed by water molecules, resulting in a defined and user- controlled temperature change.

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Normalization ID

The normalization ID is used to assign ligands to specific references in single-dose-screens.

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On-time

The term on-time is used to describe two closely related, but different aspects of a TRIC measurement.

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Pico Detection

The Dianthus NT.23 Pico optics have a higher detection sensitivity compared to standard NT.23 (Nano) optics.

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PicoDuo

Dianthus PicoDuo instruments have the great advantage of having two optical systems that can work in parallel to measure plates, thereby reducing the average time to read a plate from ~1 h to ~30 min for an on-time of 5 sec per well.

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Plate Layouts

DI.Control offers full flexibility in terms of plate layouts. Dilution series of ligands can be placed either in horizontal orientation (e.g. Well A1-A12) or vertical orientation (e.g. Well A1-H1, also see vertical plate layouts).

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Plates

The Dianthus 384-well plate has a working volume of 18-25 µL. It is made of black polystyrene material which ensures that samples are not subjected to sunlight and do not photobleach easily.

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Quality Checks

Quality checks are used in DI.Control and DI.Screening Analysis to make users aware of sample quality issues. They represent the quality controls performed on the measured data.

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Response Evaluation Mode

In DI.Screening Analysis software the on-time for analysis used in affinity screening can be selected on the data page of the software.

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Single-Dose Screening

Primary screenings are usually conducted in a single-dose setup and are best and most efficiently analyzed using DI.Screening Analysis software.

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Vertical Plate Layouts

DI.Control offers full flexibility for plate layouts. Apart from the horizontal orientation Dianthus is also able to work with vertical orientation of samples on a plate.

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Well

Dianthus 384-microwell plates contain 384 wells in 16 rows and 24 columns. Hence, it follows the SLAS standard.

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Well Categories

In DI.Control and DI.Screening Analysis, three main categories are used to assign a specific function to a certain sample. Each category is easily identified by a specific color that is always used to signify a certain category.

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Well Scans

The Dianthus instruments perform well scans in three dimensions before each well is measured for a TRIC response.

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Science


Antibodies

Antibodies are large proteins with a molecular weight of ~150 kDa. Some antibody classes such as IgMs or IgEs form multimers.

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Bromodomain Proteins

Bromodomains (BRDs) have an important role in the targeting of chromatin-modifying enzymes to specific sites that contain acetyl lysines.

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Complex

The molecular complex formed by target and ligand upon binding.

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Cooperativity

Cooperative binding are binding events in which the binding affinity of a molecule to an interaction partner is influenced by a preceding binding event.

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Disulfides

A disulfide bond, disulfide bridge, SS- bond or simply disulfide, is a functional group present in some proteins. It describes the covalent connection of two thiol groups, usually of cysteines, in the form of R–S–S–R’.

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Enzymes

Enzymes execute a wide variety of functions. For example, they regulate signal transduction via kinases and phosphatases. ATPases generate muscle contraction and active transport.

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Fluorescence

Fluorescence is the physical property of some molecules, called fluorophores, to be able to absorb a photon and emit a photon of lower energy in return.

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Fragment Based Drug Discovery

Fragment-based Drug Discovery (FBDD) has become a very popular approach in drug development.

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GPCRs

G protein-coupled receptors (GPCRs) are proteins containing seven membrane-spanning α- helices (7TMs) and form a major class of integral membrane proteins.

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His-tag

A polyhistidine-tag (His-tag) is an amino acid motif in proteins that consists of at least six histidines and is commonly positioned at the N- or C-terminus of the protein.

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Isoelectric Point

The isoelectric point (pI) is the pH at which a particular molecule carries no net electrical charge.

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Kinases

Protein kinases are enzymes that modify the function of other proteins by attaching phosphate groups to them. Since they are often involved in the regulation of the cell cycle, cell migration and cell signaling, they are popular drug targets.

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Ligand

Non-fluorescent binding partner of the target.

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Membrane Proteins

Membrane proteins interact with or are part of biological membranes and perform a variety of functions.

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Nanodiscs

Nanodiscs serve as a synthetic membrane system for the investigation of integral membrane proteins.

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Ni-NTA

Nitriloacetic acid (NTA) is a chelating agent that forms coordination compounds with metal ions. Nickel (Ni2+) is most commonly used.

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Nucleic Acids

Nucleic acids are macromolecules that store and translate the genetic information of an organism.

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Peptides

Peptides are short amino acid chains with limited or no higher-order structure. They are often used experimentally as substitutes for whole proteins since they are easy to synthesize and modify.

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Phosphatases

Phosphatases are enzymes that remove a phosphate group from its substrate.

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Photobleaching

Photobleaching describes a decay of fluorescence intensity caused by excitation light. It is typically caused by reactive oxygen species, which react with the excited states of fluorophores.

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Promiscuous Binders

The term promiscous binder in a screening context refers to small molecule ligands which display unspecfific effects toward the target molecule.

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Screening

TRIC can be used to test large numbers of interactions. Typically, a ligand library of small molecules, peptides, protein constructs or fragments (see Fragment Based Drug Discovery) is tested against one target protein, for example a kinases or receptor.

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Small Molecules

Small molecules are chemical compounds with molecular weights below 900 daltons. Most drugs are small molecules.

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Target

Fluorescent biomolecule, which binds to the ligand. The concentration of target is kept constant throughout serial dilution, while the concentration of non-fluorescent ligand is varied.

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TRIC

TRIC stands for Temperature-Related Intensity Change and is the technology powering Dianthus.

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