BioLux® Gaussia Luciferase Assay Kit

Description

Figure 1: The Photo-oxidation catalyzed by Gaussia Luciferase.

Figure 2: GLuc kinetics using the BioLux GLuc Assay Kit in either standard or stabilized assay.

Figure 3: Stability of Gaussia Luciferase at various temperatures.

Figure 4: Stability of Gaussia Luciferase at 37°C over a period of seven days.

Figure 5: Gaussia Luciferase activity after adding GLuc assay solution containing stabilizer to a sample.

Kit Components

The following reagents are supplied with this product:

Advantages and Features

Features

• Gaussia Luciferase (GLuc) possesses a natural secretory signal and upon expression is secreted into the cell medium. Therefore, lysing cells in order to assay GLuc activity is not necessary. As a result, GLuc is an ideal reporter gene for time course studies (3).
• GLuc generates over 1000-fold higher bioluminescent signal intensity, when compared to Firefly and Renilla luciferases, making it an ideal transcriptional reporter (3).
• GLuc show the highest reported activity of any characterized luciferases (4).
• The secreted protein is thermally stable (Figure 3) and has extremely high activity in light production for very sensitive assays (2). 
• The secreted GLuc is also very stable in the presence of 55µM ß-mercaptoethanol, which is typically used in culturing mouse stem cells (Figure 4).
• The GLuc-containing samples, i.e., growth media or cell lysates, can be stored at -20˚C for long-term storage or 4˚C for several days without loss of activity.
• The stabilizer component of the this assay system provides steady kinetics over a long period of time allowing users the time required for high throughput analysis as well as manually delivered assays.

Properties and Usage

Storage Temperature

Related Products

Companion Products

• Anti-GLuc Antibody
• pCMV-GLuc 2 Control Plasmid
• pSV40-GLuc Control Plasmid
• pGLuc-Basic 2 Vector
• pTK-GLuc Vector
• pGLuc Mini-TK 2 Vector
• BioLux® Cypridina Luciferase Assay Kit
• pCMV-CLuc 2 Control Plasmid
• pSV40-CLuc Control Plasmid
• pCLuc-Basic 2 Vector
• pTK-CLuc Vector
• pCLuc Mini-TK 2 Vector

Notes

1. Because of the stability of GLuc, the activity measured in the growth media of GLuc-expressing culture reflects the protein that has accumulated up to the time of sampling.
   
   
2. For the standard assay solution, i.e. solution that does not contain stabilizer, equilibration of the assay solution is not necessary.After adding the GLuc assay solution to the sample, we recommend a delay of 1-5 seconds before taking a measurement. Keeping the delay time consistent across experiments will ensure reproducibility.
   
   
3. For the stabilized assay solution, i.e., the stabilizer-containing GLuc assay solution, the solution should be equilibrated at room temperature for 25 minutes (protect from light in a tightly capped tube/bottle) before adding to the sample.
   
   After adding the equilibrated GLuc assay solution to the sample, we recommend a delay time of 35-40 seconds before taking a measurement in order to reach maximum level of detection. This is especially important when the GLuc activity level is low (e.g. < e4 RLU). For example, the readout obtained after 35-40 seconds of delay is ~e4; when compare to 30, 20 and 10 seconds of delay, the readouts are as follows: ~2% decrease (for 30 seconds of delay), ~7% decrease (for 20 seconds of delay), & ~20% decrease (for 10 seconds of delay) in RLU (refer to Figure 5).
   
   
4. Use the prepared GLuc assay solution within 24 hours. The unused portion of the assay solution should tightly capped and stored at -20˚C. It should be completely thawed (in the dark) at room temperature before use.
   
   
5. The linear range of the luminometer used for the assay must be established. This is easily done by assaying serial dilutions of a sample. In addition, the assay solution itself, as well as the conditioned media (i.e. growth media from untransfected cells) should be included in the assay to establish the background signal in the assay.
   
   
6. If excess activity for the instrument range is found, the sample should be diluted in either PBS or 10% serum-containing media. The integration time can also be reduced.
   
   
7. When assaying the serial dilution of a sample, it is best to assay the most diluted samples first and the most concentrated samples last. This will help to minimize false readings, i.e., cross-talk effect (signals from samples of high RLU cross into that of the next sample). The cross-talk effect seems to be more pronounced when plates (white or black) with clear-bottoms are used.
   
   
8. The BioLux GLuc Assay Buffer and the BioLux GLuc Stabilizer can be stored at 4˚C while the BioLux GLuc Substrate must be stored at -20˚C.

References

1. Verhaegen M. and Christopoulos T.K. (2002). Anal. Chem. 74, 4378-4385.
2. Tannous, B.A., Kim, D.E., Fernandez, J.L., Weissleder, R., and Breakefield, X.O. (2005). Mol. Ther. 11, 435-443.
3. Wu, C., Suzuki-Ogoh, C. and Ohmiya, Y. (2007). BioTechniques. 42, 290-292.
4. Goerke, A., Loening, A., et al. (2008). Metabolic Engineering. 10, 187–200.