IRDye^® 800RS Infrared Dye

IRDye 800RS is an excellent choice for nucleic acid applications.

• NHS ester reactive group for labeling primary and secondary amino groups

• Labeled nucleic acids easily purified by reverse-phase chromatography

• Good water solubility, but low salt tolerance

• More hydrophobic than IRDye 800CW

• Useful for self-quenching protease assays

• IRDye 800RS is not recommended for most protein applications, because of its potential for self-quenching

Solvent	Ext. Coeff. (M-1cm-1)	Abs Max (nm)	Em Max (nm)	Stokes Shift (nm)
Methanol	300,000	770	786	16
Water	200,000	767	786	19
1X PBS	200,000	767	786	19
PBS: Methanol	270,000	770	786	16

IRDye^® 800RS for Fluorogenic
Protease Assays

Because IRDye^® 800RS will self-quench when dye molecules are close together, fluorogenic protease substrates can be created.

Example:

If casein protein is heavily labeled with IRDye 800RS, the dye becomes self-quenched. When casein is cleaved by a protease, quenching is relieved and fluorescent signal is generated. Labeled casein can be used for general protease assays.

Advantages:

• Simple – just mix and read

• Sensitivity – near-infrared detection reduces background, light scatter and interference caused by other compounds

• Flexible – no stop solution required; easy to monitor reactions over time

• Uses less substrate than visible fluorescence kits

• Up to 20-fold fluorescence intensity enhancement upon digestion

• Can be used for protease inhibitor screening

Self Quenching and Signal Generation

In the heavily labeled substrate, IRDye 800RS fluorophores are self-quenched by interaction of dye molecules that are in close proximity.

After protease cleavage, the fluorophores are separated. Quenching is relieved and fluorescent signal is generated.