Avitag™: Biotinylated Gaussia luciferase Data Sheet
Catalog No. 0308
Product Description
Gaussia luciferase is a luminescent protein with quantum yields higher than the luciferases
from Renilla sp. and firefly. The enzyme catalyzes the oxidation decarboxylation
of coelenterazine to give light (λmax = 485nm) and coelenteramide. ATP is not needed
for activity. Gaussia luciferase is salt tolerant and retains it activity in the presence of
non-ionic detergents, cholate, and deoxycholate. In addition, the enzyme is thermal
tolerant and can retain activity at temperatures up to 60ºC. The protein is also resistant
to irreversible denaturation. Enzymatic activity can be recovered after treatment with
7M guanidine HCl or 8M urea in the presence of detergent.
Biotinylated Gaussia luciferase has a single biotin molecule covalently attached,
yielding a protein with a biotinylation peptide sequence tag. By spacing the biotinylation
site away from the protein’s active site, via the peptide sequence modification,
inactivation by direct biotinylation is prevented. The biotinylated luciferase, when
complexed with avidin or streptavidin, can be used for the detection of low levels of an
analyte. Detection of pmole quantities of an analyte have been reported.1
| Product Specifications |
| Protein |
1mg (0308-1), 250µg (0308-2) |
| Long-Term Storage |
2 years at - 80°C
Aliquot to avoid repeated freezing and thawing. |
| Short-Term Storage |
1 month at 4°C |
| Formulations |
In: 10mM Na2HPO4, 140mM NaCl, 2 mM KH2PO4, 3mM KCl,
10% glycerol, pH 7.6 |
| Molecular Weight |
25kDa by SDS- PAGE
22kDa |
Instructions for Use
| 1. |
The amount of luciferase used for a given assay should be empirically determined
by titering the enzyme. The amount of luciferase that gives the highest signal:
noise ratio should be selected.
|
| 2. |
Prepare a colenterazine stock by dissolving the powder in 1mL of acidified
methanol (50µL concentrated HCl to 10mL of anhydrous methanol) to give a
200µM solution. Aliquot and store at -80ºC for up to 4 weeks. A working solution
of coelenterazine is prepared by diluting the stock solution to 10µM in
18Mohm water.
|
| 3. |
For performing luciferase assays, dilute the enzyme in 50mM Tris-Cl, 0.5M NaCl,
pH 7.8 to the desired RFU per assay. Add coelenterazine to 2.5µM and immediately
measure the light production at 485nm with a 5-10 second integration.
|
The graph on the top shows the emission spectrum for
Gaussia luciferase (λemax= 485nm). The lower graph shows
the effects of salt concentration and pH on light emission of
Gaussia luciferase.
Material Safety Data
FOR RESEARCH USE ONLY. NOT INTENDED OR APPROVED
FOR HUMAN, DIAGNOSTICS OR VETERINARY
USE. Do not ingest, swallow or inhale. Do not get in
eyes, on skin, or on clothing. Wash thoroughly after
handling. For complete safety information see full
Material Safety Data Sheet.
References
| 1. |
Verhaegen, M. and Christopoulos, T. K. 2002. Recombinant Gaussia
Luciferase. Overexpression, Purifi cation, and Analytical Application
of a Bioluminescent Reporter for DNA Hybridization. Anal. Chem.
74: 4378-4385.
|
| 2. |
Avitag™ is a trademark of Avidity, LLC. |
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