Targeting Systems || Gaussia luciferase assay reagent

Products > Drug Discovery > Luciferase Assay Reagents > GAR-2

Show Price List

Gaussia Luciferase Assay reagent (GAR-2, Catalog ·0090A)
Stable version

Catalog no.	Size	Description	Price
GAR-2	1000 assays	Gaussia luciferase assay reagent stable version	$400.00

Please call for special pricing on bulk reagent purchase.

Product Description:

The GAR-2Assay 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 decreases the absolute value of light output slightly but 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.. Although the Gaussia luciferase signal obtained upon assaing samples with GAR-2 shows excellent stability making it suitable for HTS, we have recently developed a further improvement (GAR-2B)wherein the signal intentiy is much brighter than that obtained using the GAR-2 reagent and the signal stability is also much better.

About 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.

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

Figure 2: Stability of the GLuc bioluminescent signal using the GAR-2 reagent.
Panel A: With Stabilizer.
Panel B: Without Stabilizer
Using the GAR-2B version of the Gaussia luciferase assay reagent the bioluminescent signal remainsvery stable (Panel A). 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:

Native Gaussia Luciferase (GLuc) possesses a natural secretory signal and upon expression is efficiently secreted into the cell medium. Cell-lysis not necessary for assaying the luciferase
Gaussia Luciferase generates over 1000-fold 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.
GLuc shows the highest reported activity of any characterized luciferases (1).
The secreted protein is thermostable and has extremely high activity in light production allowing for very sensitive assays (1,2).
The secreted GLuc is also very stable in the presence of 55 μM β-mercaptoethanol, which is typically used in culturing mouse stem cells.
The GLuc-containing samples (i.e. growth media or cell lysates after transfection) can be stored at -20°C for long-term storage.

Gaussia Luciferase Assay Protocol (GAR-2)

Kit Contents:

1.50ml GAR- (Gaussia Assay reagent) Dilution Buffer Store at 4°C
8ml GAR Stabilizer- Store at 4 °C
500µl Coelenterazine substrate (100X), Store tightly capped at -20°C

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

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

Intracellular Gaussia 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)

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:

Tannous, B.A., Kim, D.E., Fernandez, J.L., Weissleder, R., and Breakefield, X.O. (2005) Mol. Ther, 11, 435-443.
Wu, C., Suzuki-Ogoh, C. and Ohmiya, Y (2007) BioTechniques, 42, 290-292.

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.