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Example: Fluorescence resonance energy transfer (FRET) protease assays:
Uncleaved Caspase-3 substrate is fluorescence-quenched by IRDye QC-1
Substrate cleavage relieves quenching and generates signal
No interference from uncleaved substrate
Advantages of IRDye® FRET protease assays
Simplicity - just mix and read
Flexibility – no stop solution required; easy to monitor reactions over time
Sensitivity – near-infrared detection reduces background, scattering and interference caused by other compounds
Sub-nanomolar enzyme detection1
60-fold fluorescence intensity enhancement upon digestion
Suitable for inhibitor and drug candidate IC50 measurement, and high throughput screening of enzyme activity
Excellent water solubility
1. Analytical Biochemistry 388 (2009) 220-28.
Fluorogenic Caspase-3 substrate with DEVD consensus sequence
A) Quenched substrate. When excitation light is absorbed by IRDye 800CW, energy is transferred to IRDye QC-1. No fluorescence is emitted.
B) Cleaved substrate. Caspase-3 cleavage of peptide allows dye and quencher to move apart, and relieves quenching.
Transcreener ADP2 and GDP FI Assays from BellBrook Labs
LI-COR is working with BellBrook Labs to create competitive immunoassay kits for high-throughput screening (HTS). Building upon the success of prior incorporation of LI-COR's IRDye® QC-1 quencher conjugate into its Transcreener® ADP2 FI Assay, BellBrook Labs has chosen to use the same quencher conjugate in its Transcreener GDP FI Assay. The Transcreener GDP FI Assay is designed specifically for HTS and uses a highly specific, proprietary antibody for GDP, meaning that it can be used with any enzyme that converts GTP to GDP. The Transcreener GDP Detection Mixture is comprised of a GDP Alexa Fluor® 594 Tracer bound to the GDP Antibody conjugated to LI-COR's IRDye® QC-1 quencher conjugate. Measurement of GDP is done with red fluorescent intensity (FI) readout.