Pierce™ Cypridina-Firefly Luciferase Dual Assay Kit

The Thermo Scientific Pierce Cypridina-Firefly Luciferase Dual Assay Kit provides the necessary reagents to simultaneously detect intracellular Cypridina and Red Firefly luciferase activity in mammalian whole cell lysates.

Features of the Cypridina-Firefly Luciferase Dual Assay Kit:

• Simultaneous—concurrent, filter-based, wavelength-separated detection of two luciferase activities
• Sensitive—measure bright blue Cypridina and red firefly luciferase activities in the same sample
• Fast—no quenching step required, unlike in traditional sequential dual assays
• Multiplex—capable of quantitating two cellular activities in the same sample(s)

The Pierce Cypridina-Firefly Luciferase Dual Assay Kit is a highly sensitive assay that allows for the simultaneous detection of Cypridina and Red Firefly luciferase activity. Cypridina luciferase acts as an experimental reporter with constitutively active red firefly as a normalization control. As shown in the animated videos below, this reporter-and-control combination enables simultaneous monitoring of experimental reporter and control luciferase activities in a single-read assay without the need for two-step addition of substrate reagents or quenching. The assay working solution contains substrates for both luciferases, and the reactions occur simultaneously with flash-type kinetics. The resulting luminescent signals are spectrally resolvable using filters. In a single sample, researchers can assay transcriptional activity of regulatory elements, signal transduction pathways, and effects of activators or inhibitors.

Includes:

Cell lysis buffer, reaction buffer, and substrates Vargulin and D-luciferin

Requires:

Cypridina and Red Firefly luciferase reporters; Filter sets in the ranges of 425 to 525nm for Cypridina Luc and 615 to 675nm for Red Firefly Luc. Luminometer or other instrument capable of monitoring luminescence, such as the Varioskan ALF or the Varioskan LUX microplate readers; Injector required for assessing more than 24 wells at a time.

Applications:

• Study two regulatory elements at the same time
• Monitor two signaling pathways simultaneously
• Enable studying more than one target per screen (e.g. off-target effects)

Application Videos:

Cypridina luciferase produces brighter bioluminescent signal than firefly and native Renilla luciferases. The bioluminescent signal (λmax=463nm) produced by Cypridina luciferase results from the oxidation of Vargulin. This reaction does not require adenosine triphosphate (ATP) or other cofactors. Red firefly luciferase is a mutant form of the Japanese firefly luciferase from Luciola cruciata. This luciferase produces a red-shifted emission spectrum (λmax= 613nm) that results from the oxidation of D-luciferin in the presence of ATP. Red firefly and Cypridina luciferases are used in the dual assay because the signals are spectrally resolvable.

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