Stable version

Catalog no.	Size	Description	Price
RLAR-1	1000 assays	Renilla luciferase assay reagent stable version	$400.00

Please call for special pricing on bulk reagent purchase.



The RLAR-1 Assay Kit includes an additional stabilizer component, as well as a modified assay buffer, which allows the use of the assay in high throughput format. With the standard protocol the light emission decays rapidly. The addition of stabilizer confers excellent signal stability (approximately 10% decay in 1 hour, Figure 2A). This three component assay system provides the user with 2 options: (a) use the assay without stabilizer for enhanced light output or (b) use with the desired amount of stabilizer for enhanced stability.

About Green–emitting Renilla Luciferase:

Green-emitting Renilla Luciferase: The emission max of the Green-emitting Renilla luciferase (527 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 the bioluminescent signal both in vitro and in vivo. Native Renilla luciferase is an intracellular enzyme and does not contain a secretory signal. The Green-emitting mutant has therefore been modified with a synthetic secretory signal at the amino terminal end to express it in a secretable form. The Green-emission maximum of this Renilla mutant offers advantages in multiplexed applications with Blue and Red-emitting luciferases. Promoter acitivty can be assessed by quantitating REnilla luciferase activity in the supernatant media or cell lysates of transfected cells.

Figure 1: Photo-oxidation of coelenterazine catalyzed by Gaussia luciferase



Figure 2: Stability of the Renilla luciferase (RLuc) bioluminescent signal using the RLAR-1 reagent. 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 decays faster than in the presence of stabilizer. Note: Data presented is average of triplicate determinations measured on a Turner TD2020 luminometer.


Kinetics of Renilla Luciferase Activity

• Native Renilla Luciferase does not possess a natural secretory signal, However, the green=emitting renilla mutant has been engineered with a secretory signal and upon expression is efficiently secreted into the cell medium. Cell-lysis is therefore not necessary for assaying the luciiferase
• The Green–emitting Renilla Luciferase is about 40 times brighter than commonly used Firefly and Renilla Luciferases (human codon optimized) when expressed in mammalian cells under the same promtoter, making it an ideal transcriptional reporter (1).
• The GLuc-containing samples (i.e. growth media or cell lysates after transfection) can be stored at -20°C for long-term storage.

Kit Components

1. 100ml RLAR- (Renilla Luciferase Assay reagent) Dilution Buffer
   Store at 4°C
2. 8ml RLAR Stabilizer
   Store at 4°C
3. 500µl Coelenterazine substrate (100X)
   Store tightly capped at -20°C

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

1. Dilute the concentrated coelenterazine (RLAR substrate), provided as a 100X formulation to 1X using the RLAR assay dilution buffer. For example to prepare 5 ml of assay reagent dilute 50 ul of 100 X coelenterazine with 4.95 ml of RLAR assay dilution buffer.
2. Pipette 20µl of Gaussia Luciferase Sample in Assay dish/tube
3. Add 4 ul or 8 µl RLAR stabilizer and also do one assay without stabilizer
4. Add 100 µl of Gaussia luciferase assay reagent (prepared as described in step 1)
5. Assay in luminometer integrate for 10 seconds (or as you wish)

Intracellular Renilla luciferase activity
Lyse cells using our lysis buffer (Catalog no 5X CLR-01). Follow cell lysis protocol supplied with the product (see below). 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.

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.

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