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EFC

Enzyme Fragment Complementation Assay Technology

DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology offers drug discovery the means to interrogate biomolecular reactions for advancing therapeutic drug and screening programs. Robust, reliable assay technology, can be used for both biochemical and cell based formats.

EFC is a homogeneous, non-radioactive detection technology based on two genetically engineered β-galactosidase fragments - a large protein fragment (Enzyme Acceptor, EA) and a small peptide fragment (Enzyme Donor, ED). Separately, the β-gal fragments are inactive, but in solution, they rapidly recombine to form active β-galactosidase enzyme that hydrolyzes substrate; producing an easily detectable chemiluminescent or fluorescent signal.

Links to the following sections:

• PathHunter® Cell-Based Assay Formats
• HitHunter® Biochemical Assay Formats

EFC Chemiluminescent Technology Advantages

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-, and 1536-well microplate applications, creating value and cost efficiency depending on the biology.

Mix and read assay protocols

Mix and read assay homogeneous protocols ensure easy adaptation for automation. EFC assays do not require washing, centrifugation or filtering.

No special instrumentation required

Use existing instrumentation, such as standard luminometers, multi-label microplate readers and CCD cameras.

Cost effective

Reproducible, low hit rates and low batch to batch variation saves reagents, reducing operating costs.

Enzyme Donor (ED), EFC label

ED is a small (4-11 kD) non-isotopic tag, which can either be chemically conjugated for biochemical, competitive immunoassays or receombinantly expressed in cells.

As a biochemical lable, ED is a small peptide that can ve covalently attached to a variety of small molecules or proteins without changing their properties and function. Becuase ED is a ribbon peptide with no tertiary structure, there are no restrictions on the size and molecule weight of the molecule that is labeled with ED.

As a recombinant label ProLabel™ or ProLink™ can be used for measuring protein:protein interactions or protein trafficking.

Enzyme Acceptor (EA), inactive EFC enzyme for detection

EA is an inactive EFC β-gal enzyme, Lacking the amino acids contained in the ED peptide. In solution, EA can rapidly recombine with either conjugated ED or expressed ProLabel ProLink fusion proteins by a process called complementation to form active EFC β-gal enzyme, EA can also be recombinantly expressed in cells without any toxic effect.

Active EFC Enzyme and Substrate for detection

β-galactosidase (E.C. 3.2.1.23) specifically and reproducibly hydrolyzes numerous substrates. Because active EFC β-gal enzyme is comparable in activity to the native β-gal enzyme. It hydrolyzes specific substrates to produce a signal detectable by a variety of microplate readers - colormetric, fluorescent intensity, luminometers, multimode readers and CCD cameras. DiscoveRx 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.