Dual Luciferase Assay Reagent for Gaussia luciferase - Red Firefly Luciferases

Catalog no.	Size	Description	Price
DLAR-1	1000 assays	Gaussia Luciferase-Red-emitting Firefly (Luciola) Luciferase Dual Assay Reagent	$850.00

Please call for special pricing on bulk reagent purchase.

Description:

DLAR-1: 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-1 dual assay reagent is based on the luciferase reporters enlisted below:

Gaussia luciferase: Gaussia Luciferase is a luciferase from the marine copepod Gaussia princeps (1,2). This luciferase, which does not require ATP, catalyzes the oxidation of the substrate coelenterazine in a reaction that produces light, and has considerable advantages over other luminescent reporter genes such as secretablity and a much brighter signal intensity iaddition to excellent stability of the bioluminescent signal (approximately 10% decay in an hour. The luminescence measured from the supernatant of cultured cells transfected with a plasmid expressing GLuc is proportional to the amount of enzyme produced, which in turn, reflects the level of transcription. Alternatively a cell lysate can be used for the assay. Although most of the activity is secreted, measurements can be made from the cell lysates as well.

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 appro priate filters.

Figure 1: Stability of the bioluminescent signal of Gaussia Luciferase (Panel B) and Firefly luciferase (Panrl A) using the DLAR-1 reagent. This reagent is useful for HTS applications involving both Gaussia 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 Gaussia luciferase 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)

Advantages:

Native Cypridina Gaussia luciferase possesses a natural secretory signal and upon expression is efficiently secreted into the cell medium. Cell-lysis not necessary for assaying the lcuiferase
Gaussia 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 emission spectra of Gaussia 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 Gaussia luciferase and Red firefly luciferase (i.e. growth media or cell lysates after transfection) can be stored at -20°C for long-term storage. (5)

Assay Protocol for DLAR-1:

Gaussia-Red Luciola luciferase dual assay reagent.

Kit Contents:

DLAR 1 (Gaussia-Firefly luciferase Dual Assay Buffer)
Store at -20°C
100x Coelenterazine - Store at -20°C
5X’CLR-1, Cell Lysis Buffer
Store at 4°C

Assay Protocol: Make sure all buffers are at room temperature prior to assay

Dilute 10 µl of 100X Gaussia luciferase substrate (coelenterazine) per 1 ml using DLAR-1 assay buffer.
Mix well by inverting several times
To 10 or 20 ul of sample, add 50 ul of DLAR -1 reagent and read in the luminometer.

Note: For reading in a plate luminometer pipette samples into wells first. Then add assay reagents as described above.

Intracellular Gaussia 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)

Dilute the 5X CLR buffer 1:5 with water.
Aspirate cell culture media and wash cells twice with serum free DMEM.
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
Shake for 20 min at 400 rpm on an orbital shaker (room temperature).
Mix 5-20 µl of luciferase containing sample or cell lysate with 50 µl of the luciferase assay reagent (DLAR-1) and read immediately in the luminometer.
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.

References:

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
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.
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.

Custom Reagents:

We can provide custom formulations to fit your HTS application. Call our tech support team at 1-866-620-4018 or email us info@targetingsystems.com or targetingsystems@gmail.com

Please check out our website 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.