Overview
LANCE™ and LANCE™ Ultra assays require a microplate reader that is capable of time-resolved fluorescence. We recommend filter-based fluorometers, though monochromator instruments can be used. Use of monochromator instruments may result in decreased assay sensitivity. We recommend using Revvity's VICTOR™ X, ViewLux™ (for high throughput), or EnVision™ Multilabel Plate Reader (view our instrument product pages). Please check that you have the correct filters for this assay. If you are using an instrument manufactured by another company, we recommend that you contact their technical support team to see if they have optimized settings, filter recommendations, etc. for a LANCE TR-FRET assay.
Use an excitation wavelength of 320 or 340 nm to excite the LANCE Europium chelate. We recommend you read this assay in dual emission mode, detecting both the emission from the Europium donor fluorophore at 615 nm, and the acceptor fluorophore (at 665 nm for APC, ULight™ dye, and Alexa Fluor 647). The raw FRET signal at 665 nm can be used to process your data. The ability to analyze the signal at 615 nm can be helpful when troubleshooting.
Recommended Instrument Settings for the LANCE cAMP Assay
| Parameter | VICTOR | EnVision | ViewLux | EnSpire™ TRF option |
|---|---|---|---|---|
| Flash Energy Area | High | n/a | n/a | |
| Flash Energy Level | 150 | 100% | 600,000 | |
| Excitation Filter | 320/340 | UV2 320 | DUG11 (UMB,AMC) | UV2 320 |
| Integrator Cap | 3 | n/a | n/a | |
| Integrator Level | 2x LANCE High Count 615 | n/a | n/a | |
| Emission Filter | 1) 615 nm | 1) 203 - Eu 615 | 1) 618/8 (Eu) | 1) 615 nm** |
Emission Filter |
2) 665 nm | 2) 205 - APC 665 | 2) 671/8 (LANCE) | 2) 665 nm** |
| Delay Time | 50 µs | 60 µs | 50 µs | 60 µs |
| Readout speed, gain and binning | n/a | n/a | Medium, High, and 2x | |
| Number of Flashes | n/a | 100 | n/a | |
| Window | 100 µs (200 µs) | 100 µs (200 µs) | 354 µs | 200 µs |
| Mirror module | n/a | 462 (D400/D630) or 412 (D400) | n/a | |
| Cycle | 2000 µs | 2000 µs | n/a |
Recommended Instrument Settings for the LANCE Ultra Kinase Assay
| Parameter | VICTOR | EnVision | ViewLux* | EnSpire™ TRF option |
|---|---|---|---|---|
| Flash Energy Area | High | n/a | n/a | |
| Flash Energy Level | 150 | 100% | 800,000 | |
| Excitation Filter | 320/340 | UV 320/340 | DUG11 (UMB, AMC) | UV2 320 |
| Integrator Cap | 2 (or 3**) | n/a | n/a | |
| Integrator Level | 2x the setting in LANCE High Count 615 label | n/a | n/a | |
| Emission Filter | 1) 615 nm | 1) 203 - Eu 615 | 1) 618/8 (Eu) | 1) 615 nm** |
| Emission Filter | 2) 665 nm | 2) 205 - APC 665 | 2) 671/8 (LANCE) | 2) 665 nm** |
| Delay Time | 50 µs | 90 µs | 50 µs | 60 µs |
| Readout speed, gain and binning | n/a | n/a | medium, high, and 2x | |
| Measurement Time | n/a | 100 (200***) flashes | 20 s exposure time | |
| Window | 100 µs (200-300 µs***) | 100 µs (200-300 µs***) | 354 µs | 200 µs |
| Mirror module | n/a | 402/412 (D400) or 452/462/662 (D400/D630) | Mirror 2 (UV dichroic) | |
| Cycle | 1000 µs | 2000 µs | n/a |
* ViewLux with flat field correction, bias correction, cosmic ray detection, excitation energy compensation
** The EnSpire Multi-Label plate reader uses monochromator-based emission. The wavelengths given represent the monochromator settings. The measurement is carried out first with one monochromator setting for the whole plate, then with the second monochromator setting in a subsequent measurement step.
*** If signal too low with 2 or 100
- Laser settings for LANCE assays on an EnVision
Delay: 50 µs (you could measure delay 10µs to get slightly improved S/B)
Window time: 100 µs (it is important not to use too long window time)
Time between flashes: 16600 µs
Focus height: 6.5 mm
For research use only. Not for use in diagnostic procedures.