Assay Capabilities & Development
Robustness, simplicity and reproducibility is the key to finding an assay suitable for high throughput screening. In terms of cost and feasibility assays that have a minimum of reaction steps are better than more complicated assays. For example Fluorescence assays that do not require multiple addition and wash steps are much preferable to assays that require many additions and washes (ELISA’s for example). Here are some of the assays that can be done on the Envision Multilabel Plate reader. Other assays that are available from Perkin Elmer that can be developed for HTS. These tend to be more expensive but may be best suited for a particular application.
We recommend that all users interested in implementing a screening project come and validate the sensitivity and reproducibility of the assay on the Envision plate reader.
Fluorescence Polarization Anistropy (FP): This is an inexpensive and robust assay that is ideally suited for HTS. It requires a purified target protein and a fluorescently labeled binding partner and works best when the target is large (protein for example) and the labeled partner is small (peptide) and in cases where the affinity of the ligand is high. The assay looks for competition between the library compound and the fluorescent ligand. No washes or additions are required for this assay. We are currently setup for FP assays for Fluorescein or Texas Red labeled ligands.
Alpha Screen: The assay works as described in the figure below and is ideal for studying protein-protein interactions. The Envision plate reader is specifically set up for high sensitivity analysis of Alpha Screen assays. No washing or addition steps are required but reagents and development can be costly. More Information
Time Resolved Fluorescence (TRF): This assay is designed to detect Fluorescent ligand binding to an immobilized target. Fluorophores with longer decay times are used in this assay and fluorescence is measured at time frames where spurious background fluorescence has decayed.
TRFRET: R-FRET is based on the proximity of a donor label (europium chelate) and an acceptor label (Allophycocyanin, APC) which have been brought together by a specific binding reaction. The excited energy of the Eu-chelate is transferred by a nonradiative resonance energy transfer mechanism to an acceptor within a short distance. Fluorescent lanthanide chelates with long excited state lifetimes are used to avoid interference caused by short-lived emission from acceptor molecules excited directly rather than by energy transfer. TR-FRET has been widely used, for example, in kinase, nuclear receptor and protein binding assays. More Information
Absorbance: Self explanatory
Cell Based Assays
At this time the Pathway Discovery Resource (PDR) does not have cell culture facilities. Investigators will be asked to bring batches of transfected cells to the facility for dispensing into plates with the Flexdrop platefiller followed by robotic transfer of compounds into plates. This is non-sterile transfer. Plates will then be incubated with compound prior to analysis in the Envision plate reader. Short incubation times are required <24h to minimize contamination.
Luminescence reporter assays: Many cell based reporter assays utilize luminescent reporters. See the attached documentation for a discussion of the advantages and disadvantages of various luminescence reporter assays. Luciferase
GFP fluorescence assays: this assay relies on detecting GFP fluorescence in cells. Before commiting to this system it is important to insure that the fluorescence from your cells can be detected in a 96 or 384 well format by the Envision plate reader.