A case study demonstrates that Varioskan Flash meets the requirements for LanthaScreen assays and can be successfully used to measure TR-FRET based assays with high-sensitivity.

Time-resolved fluorescence energy transfer (TR-FRET) assays have been developed through the integration of two individually established techniques. By coupling time-resolved fluorescence (TRF) and fluorescence resonance energy transfer (FRET) together, a fluorescent energy transfer method with exceptionally low background noise--for increased accuracy--is provided. TR-FRET is widely used within a range of biotechnology and pharmaceutical applications to study multiple target classes, including protein kinases, nuclear hormone receptors, proteases, and ubiquitinated proteins.

Plate Type	Fluorophore	Volume (µL)
Standard white 384-well	Tb-anti-GST antibody	20
	Flourescein-GST-MBP	20
Low volume white 384-well	Tb-anti-GST antibody	10
	Fluorescein-GST-MBP	10

The LanthaScreen TR-FRET assay (Invitrogen, Life Technologies Corporation) works on the principle that when suitable pairs of fluorophores are in close proximity of one another, excitation of the terbium chelate donor fluorophore results in energy transfer to the fluorescein or GFP acceptor fluorophore. The benefit here is the long fluorescence lifetime of the terbium lanthanide donor fluorochrome excited at 340 nm. This allows ratiometric measurement of acceptor fluorescence emission (FRET signal) at 520 nm over the 495 nm donor emission long after the background fluorescence has dissipated. The time-resolved measurement reduces assay interference (e.g. fluorescent compounds) and increases data quality.

click to enlarge Figure 1. Fluorescence Spectra of Terbium and Fluorescein (FRET) emission. The time-resolved spectra above illustrate energy transfer occurring when terbium (donor) and fluorescein (acceptor) are brought into proximity via biomolecular interactions. The inset shows the time-resolved spectra in the absence of energy transfer.

The performance of the Varioskan Flash from Thermo Scientific was tested using a LanthaScreen Tb-anti-GST Antibody Kit. Two types of solid white 384-well microplates were used and the assay was performed according to instructions. The fluorescein-labeled GST-MBP positive control was serially diluted from 200nM stock and the Tb-anti-GST antibody was diluted to 4 nM concentration using the LanthaScreen buffer provided with the kit.

The reactions were incubated for 60 minutes at room temperature and then measured by Varioskan Flash, which was controlled with the Thermo Scientific SkanIt Software program. The required dual emission measurements were performed using the multi-wavelength measurement option, making it possible to measure both donor and acceptor emissions easily without any additional time difference.

click to enlarge Figure 2. Calibration curves of LanthaScreen assays with standard and low volume white 384-well plates.

For both plate types, the TR-FRET ratios between the acceptor and donor emission signals were calculated and calibration curves were drawn. Theoretical assay sensitivities for both plate formats were calculated using standard IUPAC 3*SD principle and Z-prime values against positive control concentrations.

Both standard and low volume white 384-well plates perform very well in LanthaScreen assays measured using the Varioskan Flash. The calibration curves show almost identical behavior with both plate types and very similar theoretical assay sensitivities were obtained. When assay robustness is evaluated based on Z-prime values, similar behavior is noticed between the standard and low volume plates. Z-prime values are well over the required limit of 0.5 with all sample concentrations over 1 nM.

These results show that Varioskan Flash fulfills all requirements for LanthaScreen assays and can be successfully used to measure TR-FRET based assays with high-sensitivity. Measuring TR-FRET assays that require dual emission analysis can be performed easily with only one measurement step using the multi-wavelength feature of the SkanIt Software. Furthermore, these assays can also be performed with the Varioskan Flash using low total assay volume, making this combination especially useful where low sample consumption is critical.

Conclusion
When performing a TR-FRET assay, accuracy in the measurement of the fluorescence is key. As such, it needs to be detected at a high-sensitivity and with minimal background interference. One such way to measure this is with the Thermo Scientific Varioskan Flash Multimode Reader. TR-FRET assays are easily optimized using the TRF spectral scanning function along with the decay calculation option. The flexibility of the Varioskan Flash, in combination with the LanthaScreen platform enables rapid application of TR-FRET technology to multiple target classes across a wide range of application areas, such as second messenger, kinase, or hormone receptor assays.