TLAR-1
Catalog no. |
Size |
Description |
Price |
TLAR-1
(secreted) |
1000 assays |
Renilla, Luciferase, Cypridina (Vargula) Luciferase, Red Firefly (Luciola) Triple Luciferase Assay Reagent |
$1000.00 |
Product Description: TLAR-1
Single solution-based triple luciferase assay reagents save time and expense...
A panel of improved, ultrasensitive, secreted luciferase reporters have been developed in an effort to enable analysis of different promoter activities or pathways in the same group of transfected cells. This approach 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 or three separate assay reaggents. Two types of triple luciferas reporter assays are being offered based on using either Cypridina (Vargula) Luciferase or Gaussia luciferase as the third reporter in combination with a Green-emitting Renilla luciferase and a novel Red-emitting firefly luciferase.
Cypridina (Vargula) luciferase, from the marine ostracod Vargula Hilgendorfi is a secreted luciferase with an emission max of 460 nm is one of the brightest known luciferases with the highest turnover number.
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 forr improved brightness (about 40 times brighter than human codon optimized native Renilla Reniformis luciferase) and extended stability of he bioluminescent signal. This Green-emitting mutant is efficiently secreted by introducing a secretory tag at the N-terminal end. An intracellular non-secreted version of this mutant is also available.
A Red-emitting luciferase from the Italian firefly Luciola Italica. The emission wavelength of the red Luciola luciferases (3-5) is 617 nm. The robust signal of our Luciola luciferase mutants (1000 times higher signal intensity compared to native Luicola luciferase) makes them attractive for multiplexed assays.
Stability of Bioluminescent Signal and Emission Spectra
Figure 1: Kinetics of luciferase activity of different luciferase reporters using luciferase assay reagents in the TLAR-1 system: Reactions were set up to measure the kinetics of the luciferase activities of different luciferases in samples of transfected cells. Luciferase activities were measured using the TLAR luciferase assay reagents.
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 and Red Luciola luciferase is 617 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 lciferases 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, Green-emittin Renilla
luciferase and Red-emitting Luciola luciferase can be easily resolved
spectrally using appropriate filters.
- The samples containing both VLuc, Red-emitting Luciola luciferase 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.
Assay Protocol for TLAR-1:
Triple Luciferase assay reagent based on Cypridina luciferase, Green-emitting Renilla luciferase and Red-emitting Luciola luciferase.
Kit Contents:
- TLAR Buffer
(contains everything except coelenterazine and Vargulin (Cypridina luciferin)
which have to be added to the required amount of reagent just before the assay.
- 100x Cypridina luciferin Substrate
- Cypridina Substrate Dilution Buffer
- 100X Coelenterazine Substrate
Protocol:
(Assay protocol for measuring luciferase activities in cell supernatants)
- Dilute 10 µl of Vargulin substrate in 1 ml of Vargula substrate dilution buffer. *just before use.
- Add required amount of 100X coelenterazine to the thawed TLAR-1 buffer. You should add 10 ul of 100 X coelenterazine per 990 ul of TLAR-1 buffer (10 assays)
- Use 10- 20 µl of sample supernatants or lysate per assay.
- Add .90 ul of TLAR -1 reagent (prepared as above just before use)
- In the last step, add 20 µl of diluted Vargulin Substrate mix well and assay.
All assay reagents should be close to room temperature at the time of assay.
Assay protocol for measuring luciferase activities in cell lysates:
The TLAR 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 for spectrally resolving luciferase activities:
Red Luciola luciferase - 617 nm long pass filter
Green Renilla luciferase - 540 nm narrow band with filter
Cypridina luciferase - 450 nm short pass filter
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.
- 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