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Gaussia Luciferase Assay reagent (GAR-1)

Catalog no.	Size	Description	Price
GAR-1	1000 assays	Gaussia luciferase assay reagent	$375.00

Please call for special pricing on bulk reagent purchase.

Description:

The GAR-1 Assay Kit is used for measurement of Gaussia luciferase activity in a standard (non-highthroughput format wherein samples can be measured quickly in a microplate reader or regular tube luminometer but long-term stability of the bioluminescent signal is not required. For applications such as high throughput screening we have developed assay reagents (GAR-2 and GAR-2B) which provide excellent stability of the bioluminescent signal due to incorporation of stabilizers. The GAR-2B reagent also provides a brighter signal intensity of the luciferase signal.

About Gaussia Luciferase:
Gaussia Luciferase (GLuc) 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 in addition 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: Kinetics of of the GLuc bioluminescent signal using the GAR-1 reagent: Measurement of Gaussia luciferase activity in supernatants of cells (transfected with Gaussia luciferase expression vectors) using GAR-1 reagent from Targeting Systems (teal color line) and comparison with measurements obtained using the Renilla luciferase assay reagent form another commercial vendor (dark blue line).
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).
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 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-1)

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 into assay dish or luminometer tube
Add 50 µl of Gaussia luciferase assay reagent (prepared as described in step 1). Mix well
1.Assay in luminometer, integrate for 10 seconds (or as desired)

Intracellular Gaussia luciferase activity

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). Follow cell lysis protocol supplied with the product (see below). Assay as above using 5 ul to 10 ul of lysate

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 50 µl of the luciferase assay kit (GAR-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.