Catalog no. |
Size |
Description |
Price |
DLAR-4 |
1000 assays |
Cypridina Luciferase-Gaussia Luciferase Dual Assay |
$850.00 |
Please call for special pricing on bulk reagent purchase.
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. Many tdifferent intracellular and secreted dual luciferase assays reagents are offered by Targeting Systems. The Cycpridina –Gaussia luciferase assay system is particularly attractive because Cypridina luciferase and Gaussia 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 Gaussia luciferase assay stabilizer.
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. It is one of the brightest known luciferases with the highest turnover number.
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, the high sensitivity of Gaussia luciferase allows measurements from the cell lysates as well.
Stability of bioluminescent signal and emission spectra
Panel A
Panel B
Figure 2: Kinetics of luciferase activity of different luciferase reporters using luciferase assay reagents in the DLAR-4 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-4 luciferase assay reagents. Panel A: Stability of the Cypridina luciferase bioluminescent signal using the VLAR component fo the DLAR-4 dual assay system. Panel B: Stability of the GLuc bioluminescent signal using the GAR-2 reagent with Stabilizer.. This reagent is useful for HTS applications in which a large number of samples need to be assayed. In the absence of the stabilizer, the signal.intensity is a little higher initially but decays faster than in the presence of stabilizer. Note: Data presented is average of triplicate determinations measured on a Turner TD2020 luminometer.
Advantages:
- Cypridina Luciferase as well as Gaussia luciferas 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 and Gaussia luciferase
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 the Gaussia luciferase 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 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-Gaussia Luciferase Dual Assay Reagent- DLAR-4
Kit Contents:
- 100x Coelenterazine
Store at -20°C
- Gaussia luciferase assay dilution buffer - Store at 4°C
- Gaussia luciferase assay (GAR) stabilizer - Store at 4°C
- 100x Cypridina Luciferin Substrate - Store at -80°C
- Cypridina Substrate Dilution Buffer - Store at 4°C
- VLAR Buffer (Cypridina luciferase Assay buffer)- Store at 4°C
- Cypridina (Vargula) luciferase assay buffer - Store at 4°C
Protocol:
Assay for cell supernatants:
Gaussia Luciferase assay
- Dilute 100X coelenterazine to 1X with the required amount of Gaussia luciferase assay dilution buffer (you will need 50 ul of diluted reagent per assay).
- Pipette 5-20 µl of Gaussia luciferase or Cypridina luciferase-containing samples for assay into each well or luminometer tube
- Add 8 ul of GAR stabilizer to each samle (this is optional, a higher but less stable signal is obtained if you omit the stabilizer.
- Add 50 ul of the Gaussia luciferase assay reaent (prepared as described in step 1) to each sample. Mix well and read in the luminometer.
Cypridina (Vargula) Luciferase assay
- Dilute 10 µl of Vargulin substrate to 1 ml with the Cypridina (Vargula) substrate dilution buffer.
- Pipette 5-20 µl of Gaussia luciferase or Cypridina luciferase-containing samples for assay into each well or luminometer tube
- Add 40 ul of the VLAR assay bufferr to each sample.
- Add 20 ul of the diluted Cypridina luciferin substrate (Vargulin) prepared as described in step 1, to each sample. Mix well and read in the luminometer.
ASSAY OF LUCIFERASE ACTIVITY IN CELL LYSATES: The GAR or VLAR luciferase assay reagents 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.
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
- 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.
- 4)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.