Assay Technologies
Time-resolved FRET (TR-FRET)
TR-FRET is a highly preferred HTS technology which is based on lanthanide containing fluorescence donors and which unites TRF (Time-Resolved Fluorescence) and FRET (Fluorescence Resonance Energy Transfer) principles. The fluorescence emissions of lanthanides are long lived which allows for the introduction of a short delay between excitation and emission recording. Their complexing to macrocycles to form cryptates results in increased stability under screening conditions. In addition, the ratiometric measurement enables for correction of slight dispensing errors and sample interferences.
TRF reagents have been commercialized, e.g., HTRF (using lanthanide cryptates) and LANCE (using lanthanide chelates) (both by Revvity).
There are optimized reagents available for different target classes such as kinases, proteases, epigenetic modifiers, GPCRs and also for the quantification of a range of biomarkers and cytokines. In addition, for protein-protein-interaction assays, customized assays can be designed based on anti-tag antibodies. Most TRF assays can be carried out with purified proteins or cell lysates.
Luminescence
Luminescence is a highly preferred HTS technology because it is essentially background-free and thus usually gives very high signal-to-background ratios. In addition, assay interference by compounds is much less observed than with fluorescence technologies. This assay principle has been successfully adapted to the measurement of reporter genes, cell viability, ATP and cAMP levels, caspases, kinases, histone deacetylases and others.
When looking for a luciferase as reporter gene, please consider Nanoluciferase as a very bright and small enzyme. NanoLuc products have been commercialized, e.g., by Promega.
NanoLuc is also available as a split luciferase version enabling cellular protein-protein-interaction assays based on luciferase complementation. In the so-called NanoLuc Binary Technology (NanoBiT), the small subunit (SmBiT) and the large subunit (LgBiT) are expressed as fusions to target proteins. Upon interaction of the proteins, the subunits form an active NanoBiT enzyme which generates a bright signal in the presence of the NanoGlo substrate.
A high affinity version of the small subunit (= HiBiT) consisting of 11 amino acids can be used to tag your protein of interest. Complementation with LgBiT (in the assay buffer or expressed in cells) enables a reliable quantification of the target protein, for many proteins even at endogenous expression levels.
Cellular protein-protein-interaction assays can also be designed based on bioluminescence resonance energy transfer using NanoLuc as donor (NanoBRET). The second protein is expressed as a HaloTag fusion and is labeled with a cell permeable NanoBRET® 618 fluorophore.
Fluorescence intensity
Our plate readers support most commonly used fluorophores. Please consider that compounds can show autofluorescence which is most pronounced in the green spectrum. Thus, using red-shifted fluorophores is usually beneficial for compound screening.
Fluorescence polarization (FP)
FP can be used for HTS if S/B is > 3. As miniaturization of FP assays is mostly limited to 384 well format, the throughput is significantly less than for other technologies which are most often miniaturized to 1536 well format. Also for FP, red-shifted fluorophores are recommend, see above.
Absorbance
Absorbance assays are not supported due to low signal-to-background ratios.
High Content Imaging
Phenotypic cellular assays can interrogate complex, physiologically relevant cellular systems without prior knowledge of the target and hold great promise in identifying bioactive molecules from large compound collections.
For analysis of high content assays, two microscopes are integrated into the COMAS screening platforms. The ImageXpress Micro XL (Molecular Devices) is equipped with a large chip 2.9-megapixel scientific CMOS camera and has a solid-state light source. Objectives range from 4x to 40x. The system has filter sets for DAPI, FITC, SybrGold, TxRed and Cy5 mounted. In addition, a transmitted light and phase-contrast unit permits the imaging of cells without prior labelling. An environmental chamber allows for the analysis of life cells.
The second microscope is the Endeavor pro (Araceli Biosciences) that is a real gamechanger in high-throughput imaging with a standard scanning time of only 6 minutes for a whole 384-well plate. Scanning always captures the whole well (equalling ~25x) and includes four channels (DAPI, FITC, TxRed, Cy5) as well as brightfield. Due to the short scanning times, it is also fully compatible with life cells.
We use sophisticated image analysis software with offers both, standard feature extraction algorithms as well as AI-based analysis tools.