One System, Many Assays
The VaryScreen I includes pre-written protocols for: UV-Visible absorption, Fluorescence Polarization (eg. HitHunter IP3), FRET (including variations such as LANCE and VIPER), Luminescence (eg, Luciferase Reporter), ELISA, and AlphaScreen assays. A basic description of each assay type is provided below.
AlphaScreen is a bead-based assay which can be used to determine the extent in which two molecules are bound to each other. One component is covalently bound to an acceptor bead, which produces singlet oxygen upon illumination. The other component is attached to an acceptor bead that reacts with singlet oxygen to produce a specific fluorescence signal. The signal will only be produced when the beads are in close proximity which can only occur when the two component molecules are bound.
Many assays can be designed to take advantage of the specific absorbance of organic molecules that may be created or consumed in a biochemical reaction. Determination of the quantity of the absorbing substance, or analysis of kinetics of a reaction can be carried out. The Synergy 4 uses a xenon flash lamp and a monochromator for wavelength selection. It allows absorbance measurement within the range of 200 to 999 nanometers in 1 nanometer increments.
This method can be used to assess the degree of movement of a molecule after there is a change in its environment. For example, BioTek’s Predictor hERG assay places a tracer molecule at the intracellular side of the ion channel. The tracer is designed to produce a strong response to polarized light. When an ion channel inhibitor is added, the tracer is displaced, and its resulting randomized motion greatly reduces the degree of polarization that is observed.
The Enzyme-Linked ImmunoSorbent Assay is commonly used to detect the presence of an antibody or the corresponding antigen in a given sample. There are several general variations to the protocol, but typically there is a sequence of dispense and wash steps until a sandwich of binding molecules remain with an active enzyme attached to the last component. If the original molecule being assayed was present in the sample, the enzyme-linked protein will remain after the last wash step. When the appropriate enzyme substrate is then added, an obvious color change indicates a positive result.
The Synergy 4 is also capable of carrying out many reporter assays, such as beta-Galactoside, Glow and Firefly Luciferase. Luciferase reporter vectors are relatively easy to construct and transfect into plasmid DNA. Once cell extracts are prepared, measurement of luciferase activity is a good indicator of the presence of the desired gene expression.
Several key assays are frequently used in drug screening that take advantage of intermolecular transfer of fluorescense resonance energy transfer. Many kits are available that support such assays as LANCE, VIPER and other TR-FRET protocols. All of these are easily supported by the VaryScreen.