Catalog no.	Size	Description	Price
DLAR-3	1000 assays	Cypridina Luciferase-Red-emitting Firefly (Luciola) Luciferase Dual Assay Reagent	$850.00

DLAR-3: Single Solution-based Dual Luciferase Assay Reagent:
Save on costs and time in screening applications

A panel of improved ultrasensitive secreted luciferase reporters have been developed in an effort to enable analysis of different promoter activities in the same group of transfected cells. This  approach not only enables analysis of three or more pathways (responses) in the same group of cells but also enable one to study the response in real time without killing the cells since the three reporters are secreted. By choosing luciferases with different emission maxima we provide an additional advantage in that multiple luciferases can be  assayed using a single assay reagent and spectrally resolving the two luciferase activities using appropriate filters. The single-solution based DLAR-3 dual assay reagent is based on the luciferase reporters enlisted below:

• Cypridina (Vargula) luciferase: Cypridina luciferase (formerly known as Vargula Luciferase) from the marine ostracod Vargula Hilgendorfi is a secreted luciferase with an emission max of 460 nm. Cypridina luciferase, l offers the advantage of secretability and increased brightness (It is about 20-30 times brighter than the firefly luciferases)
• A Red-emitting luciferase from the Italian firefly Luciola Italica. The emission wavelength of the red Luciola luciferases (617 nm). The robust signal of our Luciola luciferase mutants (1000 times higher signal intensity compared to native Luicola luciferase) makes them attractive for single solution –based multiplexed assays as signall strength is typically diminished in single solution-based multiplexed assays wherein different luciferse asctivities are spectrally resolved using appropriate filters.

Stability of Bioluminescent Signal and Emission Spectra



Figure 1: Stability of the bioluminescent signal of Cypridina luciferase and firefly luciferase using the DLAR-3 reagent. This reagent is useful for HTS applications involving both Cypridina luciferase and the red-emitting Luciola luciferase. Note: Data presented is average of triplicate determinations measured on a Turner TD2020 luminometer.




Figure 2: Emission spectra of CYpridina and Firefly luciferases in samples of transfected cells (lysates or supernatants). The emission spectra were recorded on a Fluorolog-3 spectrofluorometer (Horiba Scientific, Japan) using a liquid nitrogen cooled CCD. The luciferases were assayed by mixing 200 ul of the sample with the appropriate luciferase assay reagent to obtain spectral profiles. Emission max of Cypridina Luciferase is 463 nm; Red italica 617 nm. (Data courtesy of Justin Rosenberg, Dr Bruce Branchini's lab, Connecticut College, USA)

• Native Cypridina (Vargula) Luciferase (VLuc) possesses a natural secretory signal and upon expression is efficiently secreted into the cell medium. Cell-lysis not necessary for assaying the luciferase
• Cypridinaia Luciferase is one of the brightest known lciferases with the highest turnover number and a higher bioluminescent signal intensity, than commonly used Firefly and Renilla Luciferases, making it an ideal transcriptional reporter (1).
• The stabilizer component of this assay system provides steady kinetics over a longer time period allowing users the time required for high-throughput analysis as well as manually delivered assays.
• The secreted protein is stable and has extremely high activity in light production allowing for very sensitive assays (1,2).
• The emission spectra of Cypridina luciferase and the red-emitting Luciola luciferase show very little overlap and therefore can be easily resolved spectrally using appropriate filters (3)
• The samples containing both VLuc and Red firefly luciferase (i.e. growth media or cell lysates after transfection) can be stored at -20°C for long-term storage. (5)

Cypridina-Red Luciola luciferase dual assay reagent.


Kit Contents:

1. DLAR 3 (Firefly/Cypridina Dual Assay Buffer). Store at -20°C
2. 100x Cypridina Luciferase Substrate - Store at -80°C
3. Cypridina Substrate Dilution Buffer - Store at 4°C
4. 100x Coelenterazine - Store at -20°C

Protocol

1. Dilute 10 µl of Vargulin substrate in 1 ml of Vargula substrate dilution buffer. *
2. Mix well by inverting several times.
3. To 10 or 20 ul of sample, add 90 ul of DLAR -3 buffer and 20 ul of diluted Cypridina luciferin. Mix well and read in the luminometer.

Note: For reading in a plate luminometer pipette samples into wells first. Then add assay reagents as described above. If the samples are likely to be read soon then the diluted Cypridina luciferin can be mixed with the DLAR-3 buffer and the mixture can be added instead of adding the diluted Cypridina luciferin separately. However, the end user must make sure that this format gives comparable readings to those obtained when the diluted Cypridina substrate is added separately.


Intracellular luciferase activity
Lyse cells using our lysis buffer (Catalog no 5X CLR-01). Follow cell lysis protocol supplied with the product. Assay as above using 5 ul to 10 ul of lysate

NOTE: If you need to measure intracellular luciferase activity, lyse cells first using the cell-lysis buffer from Targeting Systems. (catalog no 5X CLR-01)

1. Dilute the 5X CLR buffer 1:5 with water.
2. Aspirate cell culture media and wash cells twice with serum free DMEM.
3. Add enough of 1X cell lysis buffer to cover cells. Add enough lysis buffer to cover cell.s (50 ul for 96-well, 300 ul for a 12-well, 800 ul for a 6-well dish and 3 mll for a 10 cm dish
4. Shake for 20 min at 400 rpm on an orbital shaker (room temperature).
5. Mix 5-20 µl of luciferase containing sample or cell lysate with 100 µl of the luciferase assay kit (TS-1) and read immediately in the luminometer.
6. All assay reagents should be close to room temperature at the time of assay.

Filter Selection:
We recommend a 575 nm long pass filter to read the red-emitting firefly luciferase and a 525 nm short pass filter to read Cypridina luciferase.


We can provide custom formulations to fit your HTS application.
Call our tech support team at 1-866-620-4018 or email us. Please check out our webstie www.targetingsystems.net for novel luciferase –based multiplexed assays which enable analysis of up to four promoter activities in the same group of transfected cells.


Relevant References:

1. Thompson, E. M., Nagata, S., and Tsuji, F. I. (1990) Vargula hilgendorfii luciferase: a secreted reporter enzyme for monitoring gene expression in mammalian cells Gene (Amst.) 96, 257–262
2. Shin-ya Nishide, Sato Honma, Yoshihiro Nakajima, Masaaki Ikeda, Kenkichi Baba, Yoshihiro Ohmiya, and Ken-ichi Honma (2006) New reporter system for Per1 and Bmal1 expressions revealed self-sustained circadian rhythms in peripheral tissues. Genes Cells, Oct 2006; 11: 1173 - 1182.
3. Elisa Michelini, Luca Cevenini, Laura Mezzanotte, Danielle Ablamsky, Tara Southworth, Bruce Branchini, and Aldo Roda* (2007) Spectral-Resolved Gene Technology for Multiplexed Bioluminescence and High-Content Screening. Anal. Chem., 10.1021/ac7016579 S0003-2700(70)01657-8
4. BR Branchini, TL Southworth, JP DeAngelis, A Roda, and E Michelini (2006) Luciferase from the Italian firefly Luciola italica: molecular cloning and expression. Comp Biochem Physiol B Biochem Mol Biol, Oct 2006; 145(2): 159-67.
5. BR Branchini, DM Ablamsky, MH Murtiashaw, L Uzasci, H Fraga, and TL Southworth (2007) Thermostable red and green light-producing firefly luciferase mutants for bioluminescent reporter applications. Anal Biochem, Feb 2007: 361(2): 253-62.

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