Catalog no. |
Size |
Description |
Price |
DLAR-5 |
1000 assays |
Renilla luciferase
Cypridina Luciferase Dual Assay |
$850.00 |
Product Description:
Single Solution-based Dual Luciferase Assay Reagents:
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 two different promoter activities in the same group of transfected cells. This approach also enables 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.. Many tdifferent intracellular and secreted dual luciferase assays reagents are offered by Targeting Systems. The Cycpridina –Renilla luciferase assay system is particularly attractive because Cypridina luciferase and Renilla lucifersae both have robust activites both in the intracellular as well as secreted fraction and both have stable bioluminescent signals when assyed with this reagent .
The stability of the Renilla luciferase bioluminescent signal (Fig. 2) can be further enhanced by adding a Renilla luciferase assay stabilizer.
- Cypridina (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)
- Green-emitting Renilla Luciferase: The emission max of the Green-emitting Renilla luciferase (530 nm) makes it ideal for multiplexed assays with blue and red emitting luciferases. This luciferase has been engirneered for improved brightness (about 40 times brighter than human codon optimized native Renilla Reniformis luciferase) and extended stability of he bioluminescent signal
Stability of bioluminescent signal and emission spectra
Panel A
Panel B
Figure 1: Kinetics of luciferase activity of different luciferase reporters using luciferase assay reagents in the DLAR-5 system: Reactions were set up to measure the kinetics of the luciferase activities of different luciferases in samples of transfected cells. Luciferase activities were assayed using the DLAR-5 luciferase assay reagents. The decay of the renilla luciferase signal shown in Panel B above can be greatly minimized (ie the bioluminescent signal can be rendered much more stable by addition of a Renilla luciferase stabilizer to thes DLAR-5 buffer.
Figure 2: Emission spectra of different luciferases in samples of transfected cell lysates. Relative luciferase activities of Cypridina, Renilla and Red Luciola luciferases were assayed with the appropriate luciferase assay reagent to obtain spectral profiles. The emission max of Vargula luciferase is 463 nm; Green Renilla luciferase is 527 nm .
(Data courtesy of Justin Rosenberg, Dr Bruce Branchini´s lab, Connecticut College, USA)
Advantages:
- Cypridina Luciferase as well as the improved secreted Green Renilla
Luciferase have very robust signals in both supernatants and
lysates of transfected cells. Therefore can be used as a dual
secreted reporters or dual intracellular reporters.
- 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 luciferases with
the highest turnover number and a higher bioluminescent signal
intensity, than commonly used Firefly 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 Cypridina luciferas 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 Green Renilla
luciferase can be easily resolved spectrally using appropriate filters.
- The samples containing both VLuc and secreted Green-emitting
Renilla luciferase (i.e. growth media or cell lysates after transfection)
can be stored a -20°C for long-term storage.
Cypridina-Renilla Luciferase Dual Assay Reagent- DLAR-5
Kit Contents:
- DLAR 5/Cypridina-Renilla Dual Assay Reagent) Buffer - Store at -20°C
- 100x Cypridina Luciferin Substrate - Store at -80°C
- Cypridina Substrate Dilution Buffer - Store at 4°C
- 100x Coelenterazine - Store at -20°C
Protocol
Assay for cell supernatants:
- Dilute 100X coelenterazine to 1X with the required amount of DLAR-5 assay buffer (90 ul per assay).
- Dilute 10 µl of Vargulin substrate to 1 ml with the Cypridina (Vargula) substrate dilution buffer.
- Use 5-20 µl of Renilla and Cypridina luciferase-containing samples for assay
- Assay using 90 µl of the DLAR-5 assay buffer containing 1X coelenterazine.
- As the last step, add 20 µl of diluted Vargulin Substrate buffer.
ASSAY OF LUCIFERASE ACTIVITY IN CELL LYSATES:
The RLAR luciferase assay reagent can also be used to measure luciferase activity in pre-lysed cells. 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 100 µl of the
luciferase assay kit (TS-1) and read immediately in the luminometer.
- All assay reagents should be close to room temperature at the time of
assay.
Suggested Filters: We suggest using a 430 nm short pass filter to measure Cyrpidina luciferase activity and a 540 nm log pass filter to measure the Green Renilla Luciferase signal
Relevant References:
- Yamagishi, K., Enomoto, T. and Ohmiya, Y. (2006) Anal. Biochemistry,
354, 15-21.
- Wu, C., Suzuki-Ogoh, C. and Ohmiya, Y. (2007) Biotechniques, 42,
290-292.
- 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.
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.
More citations on the Luciola, Gaussia and Cypridina and Renilla luciferases/luciferase assays are listed in the Luciferase section of citations link on our website www.targetingsystems.net
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.
Questions?
Please call our tech support 1-866-620-4018 or email us
info@targetingsystems.net