ALPHA-screen (Amplified Luminescence Proximity Homogeneous Assay) is a homogeneous luminescence based proximity assay for the analysis of protein-protein interactions. One interaction partner is attached to donorbeads (e.g. Streptavidin), while the other one is coupled to acceptorbeads (e.g. Ni-chelate).A photosensitizer compound is embedded into the donor bead. Upon illumination with laser light, ambient oxygen is converted to energy-rich, short-lived singlet oxygen by the photosensitizer compound. Within its 4 µsec half-life, singlet oxygen can diffuse approximately 200 nm in solution. Thus, when no acceptor bead is in proximity, the singlet oxygen decays without producing a signal. If donor and acceptor bead are brought together by the biological interaction of the attached biomolecules, the singlet oxygen initiates a luminescence / fluorescence cascade in the acceptor bead, leading to a signal in the 520-620 nm range.
ALPHA-screen displays both high sensitivity and low background, making it an ideal technique in high throughput screening. The high sensitivity of ALPHA-screen is based on a cascade reaction, which occurs due to the high concentration of photosensitizer inside the donor beads and the high density of fluorophores inside the acceptor beads. Upon excitation at 680 nm, each donor bead can release up to 60,000 singlet oxygen molecules per second, resulting in a very high signal amplification. The low background is maintained because autofluorescence of compounds and plates is eliminated by two mechanisms: first, bead emission is measured in a time-resolved mode (20 msec delay between excitation and detection cycles). Second, the wavelength at which the signal is read is lower than the excitation wavelength.
High Content Imaging - Fluorescence intensity and FRET
Phenotypic cellular assays can interrogate complex, physiologically relevant cellular systems without prior knowledge of the targetand hold great promise in identifying bioactive molecules from large compound collections.
For analysis of high content assays, the COMAS lab has installed an ImageXpress Micro XL (Molecular Devices). The ImageXpress Micro XL is equipped with a large chip 2.9 megapixel scientific CMOS camera and has a solid state light source. Objectives range from 4 x to 40 x. The system has filter sets for DAPI, FITC, TRITC and Cy5 mounted. In addition, a transmitted light and phase-contrast unit permits the imaging of cells without prior labelling.
A microplate mover (Orbitor RS; Thermo Scientific) is used for the automated loading of the High Content Reader on a 24/7 basis.
HTRF (Homogeneous Time Resolved Fluorescence) is a technology based on TR-FRET. TR-FRET unites TRF (Time-Resolved Fluorescence) and FRET (Fluorescence Resonance Energy Transfer) principles. This combination brings together the low background benefits of TRF with the homogeneous assay format of FRET. However, HTRF displays a unique TR-FRET chemistry, including the use of Europium and Terbium cryptate chemistries. The lanthanides have a long life and their complexing to macrocycles to form cryptates allows increased stability. Finally, the use of a patented ratiometric measurement enables correction for quenching and sample interferences.
The Envision plate reader is suitable for the readout of luminescent signals from all kinds of luciferases and luminogenic substrates. 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.
The bioluminescence resonance energy transfer (BRET) method is based on resonance energy
transfer between a light-emitting enzyme and a fluorescent acceptor. The energy donor is aluciferase, which emits light in the presence of its corresponding substrate. The energy acceptor is a fluorophore, typically a fluorescent protein, which absorbs light at a given wavelength and emits light at a longer wavelength. To investigate protein- protein interactions by BRET, one protein is fused to the donor and the other to the acceptor. If the two fusion proteins do not interact, only light emission by the luciferase can be monitored. If the two fusion proteins interact and the distance between the energy donor and acceptor is less than 10 nm, a resonance energy transfer occurs and an additional light signal corresponding to the acceptor emission can be detected.
The first variant of the original BRET assay has been called BRET2 and uses coelenterazine 400a, a chemical derivative of coelenterazine h, that shifts the maximal light emission to 395 nm. Appropriate acceptors are GFP2 and GFP10 with excitation and emission maxima of 400 and 510 nm, respectively. The major advantage of BRET2 over BRET1 method lies in the better spectral separation of the donor and acceptor emission peaks.