EFC is a homogeneous, non-radioactive detection technology based on two genetically engineered ß-galactosidase fragments - a large protein fragment (Enzyme Acceptor, EA) and a small peptide fragment (Enzyme Donor, ED). Separately, the ß-gal fragments are inactive, but in solution, they rapidly recombine to form active ß-galactosidase enzyme that hydrolyzes substrate to produce an easily detectable chemiluminescent or fluorescent signal.
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Robust, reproducible signal:
EFC is an enzymatically amplified signal, resulting in large signal to background ratios and high precision with Z' factors > 0.7.
Minimize interference:
The chemiluminescent signal minimizes interference from library compounds, introducing no artificial signal due to non-specific binding of beads or secondary labels.
Flexible:
Scaleable protocols for 96-, 384-, 384 LV and 1536- well microplate applications, creating value and cost efficiency depending on the biology.
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Mix and read assay protocols:
Add and mix assay protocols ensure easy adaptation for automation. EFC assays do not require washing, centrifugation or filtering.
Convenient:
Use existing instrumentation, such as liquid handlers either in batch or HTS mode with a wide range of luminometers, mutli-label microplate readers and CCD cameras.
Cost effective:
Reproducible low hit rates and low batch to batch variation saves reagents, reducing operating costs.
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EFC chemiluminescent detection is based on two key components - Enzyme Donor (ED) and Enzyme Acceptor (EA). Separately, these fragments are inactive, but upon mixing they spontaneously recombine to form active EFC ß-galactosidase enzyme, which hydrolyzes a substrate that can be detected by a microplate reader.
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Enzyme Donor (ED), EFC label
ED is a small (4-11 kD) non-isotopic, non-bead based label in the EFC assay system. It can either be chemically conjugated for biochemical in vitro based competitive immunoassays or recombinantly expressed in cells as a ProLabel fusion protein for measuring translocation and protein expression. When expressed alone, ProLabel is unstable and is rapidly degraded, which reduces background significantly.
As a label, ED is a small peptide that can be covalently attached or expressed with a variety of small molecules or proteins without changing their properties and function. Because ED is a ribbon peptide with no tertiary structure, there are no restrictions on the size and molecular weight of the molecule that is labeled with ED.
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Enzyme Acceptor (EA), inactive EFC enzyme for detection
EA is an inactive EFC ß-gal enzyme. It is inactive because it lacks amino acids contained in the ED peptide. In solution, EA can rapidly recombine with either conjugated ED or expressed ProLabel fusion proteins by a process called complementation to form active EFC ß-gal enzyme.
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Active EFC Enzyme and Substrate for detection
ß-galactosidase (E.C. 3.2.1.23) specifically and reproducibly hydrolyzes numerous substrates. Because active EFC ß-gal enzyme is comparable in activity to the native ß-gal enzyme, it hydrolyzes specific substrates to produce a signal detectable by a variety of microplate readers - colorimetric, fluorescent intensity, luminometers, multimode readers and CCD cameras. EFC assays and EFC related solutions are preferentially developed using a chemiluminescent substrate which produces a high intensity signal with low background that is not affected by naturally fluorescent compounds.
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Drug discovery research requires that assay technologies perform reproducibly and robustly and can be simply run on existing liquid handling and detection instrumentation. Because ED is a versatile label, the EFC technology has been adapted to accommodate a variety of biomolecular targets using biochemical in vitro assay formats and whole-cell in vivo assay formats.
The biochemical in vitro assays are formatted as competitive immunoassays, where analytes are detected in solution, without separation or wash steps, providing improved sensitivity and precision compared to conventional immunoassays. In the biochemical assays, ED is conjugated to ligand of interest (eg. cAMP, cortisol, or phosphorylated kinase substrate) which competes against free ligand for binding to a binding protein. Depending on the ligand of interest, the binding protein can be an antibody, an enzyme, or a receptor.
In the absence of free ligand, ED-conjugates are captured by the binding protein and are unavailable for complementation, resulting in low signal. In the presence of free ligand, binding protein sites are occupied, leaving ED-conjugate free to complement with EA, forming active EFC -gal enzyme for substrate hydrolysis to produce a detectable signal. A positive signal is generated in direct proportion to the amount of free ligand bound by the binding protein.
Biochemical in vitro assays are referred to as HitHunter™ assays and have been developed for the following target applications:
- GPCR and Second Messenger signaling
- Kinase activity and binding
- Nuclear hormone receptor binding
- Protease activity
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EFC detection can be adapted in vivo using an expression vector for cloning a gene of interest that is then transfected into a cell line to express ED as a fusion protein, also termed ProLabel. Lysates from cell lines expressing ProLabel fusion proteins are measured using EFC detection by adding EA and substrate.
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PathHunter bridges the gap between High Throughput Screening (HTS) and High Content Analysis (HCA), by applying EFC complementation into a whole cell in vivo detection system. The PathHunter product family is a whole-cell system that utilizes positional complementation within the context of the living cell to report target translocation.
Both EA and ED can be co-expressed in a cell to measure nuclear translocation. EA is specifically localized to the nucleus using engineered NLS motifs, while ProLabel fusion proteins maintain the natural cytoplasmic localization of the target protein. Positional Complementation occurs only when ProLabel fusion proteins translocate to the nucleus in response to extracellular stimuli. Thus PathHunter™ EFC technology provides the means to interrogate cell pathways resulting in translocation. Since PathHunter EFC assays do not require imaging technology, they are amenable to all microtiter plate formats for higher throughput, whole cell assays using simple liquid handling and microplate readers that already exist in the discovery research programs.
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Whole-cell assays and targets utilizing EFC technology are referred to as PathHunter™ and can currently cover the following applications:
- Protein Translocation
- Protein Expression
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EFC
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