HTRF assays from Cisbio have advanced to cover diverse areas, including G protein-coupled receptors, kinases and cell signaling, epigenetics, and biomarkers. A broad range of toolbox reagents and kits is offered to support studies on protein-protein interactions, nuclear receptor assays, receptor dimerization, ligand binding, receptor internalization, enzyme assays, and other applications.
HTRF uses two fluorophores, a donor and an acceptor, to label proteins or other targeted biomolecules. When these biomolecules interact, their proximity enables fluorescence resonance energy transfer (FRET) from the donor to the acceptor fluorophore.
The use of long-lifetime fluorescent lanthanide cryptates as donors enables time-resolved detection of HTRF assays, reducing short-lived background fluorescence from compounds and other materials. Detecting time-resolved FRET (TR-FRET) using a microplate reader provides a readout of biomolecular binding.
A microplate reader must meet Cisbio’s performance standards to achieve reliable HTRF results. Key factors include the reader’s optics, energy source, detectors, and detection settings.
Cisbio readers use tailored components for HTRF detection, such as a dedicated detection cartridge for the SpectraMax i3x and Paradigm readers, and a TRF module with verified filters for the SpectraMax iD5.
Cisbio’s HTRF certification program ensures that readers displaying the “HTRF compatible” logo fulfill the necessary requirements for the best HTRF performance. This article outlines the optimized settings verified by Cisbio for Molecular Devices HTRF-compatible microplate readers.
Molecular Devices HTRF-compatible microplate readers
- SpectraMax® i3x Multi-Mode Microplate Reader
- HTRF Detection Cartridge (Molecular Devices P/N 0200-7011, or custom TR-FRET for Tb/green)
- SpectraMax® iD5 Multi-Mode Microplate Reader
- HTRF Detection System (Molecular Devices P/N 6590-0144; includes Enhanced TRF Module, excitation and emission filters)
- SpectraMax® Paradigm® Multi-Mode Microplate Reader
- HTRF Detection Cartridge (Molecular Devices P/N 0200-7011, or custom TR-FRET for Tb/green)
- SpectraMax® M5e Multi-Mode Microplate Reader
- FlexStation® 3 Multi-Mode Microplate Reader
Figure 1. HTRF-compatible SpectraMax Multi-Mode Readers. SpectraMax i3/i3x and Paradigm readers can be equipped with the HTRF detection cartridge. The SpectraMax iD5 reader uses the HTRF detection system, which includes an Enhanced TRF Module and filters for excitation and emission. Image Credit: Molecular Devices UK Ltd
About the reader setup recommendations
HTRF assays are offered in three distinct donor/acceptor fluorophore combinations, each necessitating specific reader configurations to ensure optimal results:
- Europium cryptate donor with red acceptor (Eu/red)
- Terbium cryptate donor with red acceptor (Tb/red)
- Terbium cryptate donor with green acceptor (Tb/green), Tag-lite
SpectraMax i3/i3x readers
Source: Molecular Devices UK Ltd
| Parameters |
Settings |
|
| Europium cryptate donor / red acceptor, Terbium cryptate donor / red acceptor |
| Optical configuration |
Cartridge |
HTRF |
| Read mode |
TR-FRET |
| Read type |
Endpoint |
| Wavelengths |
Fixed per cartridge |
Ex 340, Em1 616, Em2 665 |
| Plate type |
User selectable |
A white plate is required for high-performance HTRF measurement |
| Read area |
User selectable |
| PMT and optics |
| Number of pulses |
30 |
| Excitation time (fixed) |
0.05 (fixed) |
| Measurement delay |
0.03 ms |
| Integration time |
0.4 ms |
| Read height |
Plate format- and volume-dependent; optimize for each new plate type or volume used |
| Terbium cryptate donor / green acceptor, Tag-lite |
| Optical configuration |
Cartridge |
Custom TR-FRET (Tag-lite HTRF) |
| Read mode |
TR-FRET |
| Read type |
Endpoint |
| Wavelengths |
Fixed per cartridge |
Ex 340/80, Em1 616/15, Em2 665/10 |
| Plate type |
User selectable |
A white plate is required for high-performance HTRF measurement |
| Read area |
User selectable |
| PMT and optics |
| Number of pulses |
30 |
| Excitation time (fixed) |
0.05 (fixed) |
| Measurement delay |
0.03 ms |
| Integration time |
0.4 ms |
| Read height |
Plate format- and volume-dependent; optimize for each new plate type or volume used |
SpectraMax Paradigm reader
Source: Molecular Devices UK Ltd
| Parameters |
Settings |
|
| Europium cryptate donor / red acceptor, Terbium cryptate donor / red acceptor |
| Cartridge |
HTRF |
| Read mode |
TR-FRET |
| Read type |
Endpoint |
| Wavelengths |
Fixed per cartridge |
Ex 340, Em1 616, Em2 665 |
| Plate type |
User selectable |
| Read area |
User selectable |
| PMT and optics |
| Number of pulses |
30 |
| Excitation time (fixed) |
0.05 (fixed) |
| Measurement delay |
0.03 ms |
| Integration time |
0.5 ms |
| Read height |
Plate format- and volume-dependent; optimize for each new plate type or volume used |
| Terbium cryptate donor / green acceptor, Tag-lite |
| Cartridge |
Custom TR-FRET (Tag-lite HTRF) |
| Read mode |
TR-FRET |
| Read type |
Endpoint |
| Wavelengths |
Fixed per cartridge |
Ex 340/80, Em1 616/15, Em2 665/10 |
| Plate type |
User selectable |
|
| Read area |
User selectable |
| PMT and optics |
| Number of pulses |
30 |
| Excitation time (fixed) |
0.05 (fixed) |
| Measurement delay |
0.03 ms |
| Integration time |
0.5 ms |
| Read height |
Plate format- and volume-dependent; optimize for each new plate type or volume used |
SpectraMax iD5 reader
Source: Molecular Devices UK Ltd
| Parameters |
Settings |
|
| Europium cryptate donor / red acceptor, Terbium cryptate donor / red acceptor |
| Read mode |
TR-FRET |
The enhanced TRF module must be installed |
| Read type |
Endpoint |
| Wavelengths |
Use filters |
Ex 340/70
Em1 665/10
Em2 616/10 |
| Plate type |
User selectable |
| Read area |
User selectable |
| PMT and optics |
| Flashes per read |
50 |
| Excitation time (fixed) |
0.05 (fixed) |
| Measurement delay |
0.1 ms |
| Integration time |
0.6 ms |
| Read height |
Plate format- and volume-dependent; optimize for each new plate type or volume used |
SpectraMax M5e and FlexStation 3 readers
Source: Molecular Devices UK Ltd
| Parameters |
Settings |
|
| Europium cryptate donor / red acceptor |
| Read mode |
TR-FRET |
| Read type |
Endpoint |
| Wavelengths |
Number of wavelength pairs: 2 |
Ex 314; Em1 620, with 570 cutoff; Em2 665, with 630 cutoff |
| Plate type |
User selectable |
A white plate is required for high-performance HTRF measurement |
| Read area |
User selectable |
| TRF settings |
Integration delay
Integration time
|
50
300
|
| PMT and optics |
Flashes per read |
50 to 100 |
| Terbium cryptate donor / red acceptor |
| Read mode |
TR-FRET |
| Read type |
Endpoint |
| Wavelengths |
Number of wavelength pairs: 2 |
Ex 340; Em1 620, with 570 cutoff; Em2 665, with 630 cutoff |
| Plate type |
User selectable |
A white plate is required for high-performance HTRF measurement |
| Read area |
User selectable |
| TRF settings |
Integration delay
Integration time
|
50
300
|
| PMT and optics |
Flashes per read |
50 to 100 |
| Terbium cryptate donor / Green acceptor, Tag-lite |
| Read mode |
TR-FRET |
| Read type |
Endpoint |
| Wavelengths |
Number of wavelength pairs: 2 |
Ex 340; Em1 520, with 495 cutoff; Em2 620, with 570 cutoff |
| Plate type |
User selectable |
A white plate is required for high-performance HTRF measurement |
| Read area |
User selectable |
|
| TRF settings |
Integration delay
Integration time
|
50
300
|
| PMT and optics |
Flashes per read |
50 to 100 |
Conclusion
Using its standard certification methods, Cisbio evaluated SpectraMax multi-mode microplate readers with time-resolved fluorescence detection. The readers exceeded HTRF performance requirements and are supplied with optimized cartridges and filters to support the full range of assays, including Tag-lite.
Acknowledgements
Produced from materials originally authored by Cathy Olsen, Sr. Applications Scientist at Molecular Devices.
About Molecular Devices UK Ltd
Molecular Devices is one of the world’s leading providers of high-performance bioanalytical measurement systems, software, and consumables for life science research, pharmaceutical, and biotherapeutic development. Included within a broad product portfolio are platforms for high-throughput screening, genomic and cellular analysis, colony selection, and microplate detection. These leading-edge products enable scientists to improve productivity and effectiveness, ultimately accelerating research and the discovery of new therapeutics. Molecular Devices is committed to the continual development of innovative solutions for life science applications. The company is headquartered in Silicon Valley, California, with offices around the globe. For more information, please visit www.moleculardevices.com.
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