Recombinant Luciferase (firefly) Protein

SDS-Page: Recombinant Luciferase (firefly) Protein [NBP3-18743] - 10% SDS-PAGE analysis

Functional: Recombinant Luciferase (firefly) Protein [NBP3-18743] - An ATP dose response curve was performed in 50 ul reaction cocktails containing decreasing amounts of ATP, 0.2 ug Luciferase, and 15 nmol luciferin in a Tris-glycine buffer, pH 7.6 supplemented with 0.5 mM EDTA, 5 mM MgSO4 and 0.1% BSA. Luminescence was measured at 22C for 10 s in quadruplicates, using a GloMax 20/20 Luminometer.

Recombinant Luciferase (Firefly) Protein [NBP3-18743]—ATP is hydrolyzed, and light is emitted when firefly luciferase catalyzes the oxidation of D-luciferin. Reaction (1) is reversible, and the equilibrium lies far to the right. Reaction (2) is essentially irreversible. When ATP is the limiting reagent, the light emitted is proportional to the ATP present.

• Applications: Func, PAGE
• Concentration: LYOPH

Recombinant Luciferase (firefly) Protein Summary

• Description: Luciferase from Photinus pyralis (firefly) recombinant, expressed in Escherichia coli, lyophilized powder, >= 10 x 10^10 units/mg protein
• Preparation Method: Luciferase from Photinus pyralis (firefly) recombinant, expressed in Escherichia coli
• Source: E. coli
• Protein/Peptide Type: Full Length Recombinant Protein
• Purity: >98%, by SDS-PAGE

Applications/Dilutions

• Dilutions:
  • Functional
  • SDS-Page
• Application Notes: Application: diagnostic assay manufacturing
  
  Specific Activity: >= 10 x 10^10 light units/mg protein
  
  Unit definition: One luciferase enzyme unit will produce one Relative Light Unit (RLU) at 20-25C over a 10 s period, measured in 100 ml assay mixture containing 40 pmole ATP and 15 nmole luciferin in Tris-glycine buffer, pH 7.6, using a GloMax(R) 20/20 Luminometer.
  
  Unit Definition Conversion Factor: There are approximately 9000 Relative Light Units (RLU) per one traditional Light Unit that uses a peak height equivalent to 0.02 uCi of 14C in a PPO/POPOP cocktail
• Theoretical MW: 62 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Packaging, Storage & Formulations

• Storage: Store at -20 degrees C. Avoid freeze/thaw cycles.
• Buffer: Lyophilized powder containing HEPES, pH 7.5, NaCl, MgCl2, EDTA, DTT and a carbohydrate stabilizer.
• Preservative: No Preservative
• Concentration: LYOPH
• Purity: >98%, by SDS-PAGE
• Reconstitution Instructions: To obtain maximal solubility it is important to reconstitute the enzyme at a high salt concentration, such as 1 M Tris buffer with any counter ion at pH 7-8. Can be prepared at a concentration of up to 5 mg protein/ml. Do not vortex and avoid agitation.

Notes

Store the product at -20C. After reconstitution, the enzyme solutions can kept at 4-8C for up to 2 days or frozen in working aliquots at -20C for at least one month. Repeated freezing and thawing is not recommended. Do not store in a frost-free freezer.

Alternate Names for Recombinant Luciferase (firefly) Protein

• firefly luciferase

Background

Luciferase is a generic term for a group of oxidative enzymes used in bioluminescence. Firefly (Photinus pyralis) and bacterial luciferase enzymes are commonly used in assay systems such as cell viability assays, reporter gene assays, and for in vivo imaging. Bacterial luciferases are flavoenzymes composed of two subunits each encoded by the luxA and luxB genes, while the firefly luciferase is a single polypeptide specified by the luc gene (1). Firefly luciferase (theoretical molecular weight: 61 kDa) oxidizes the substrate luciferin to oxyluciferin in a bioluminescent reaction requiring Mg2+ and ATP (2,3). This reaction produces a flash of yellow-green light with an emission peak around 560nm that can be detected by a luminometer (3). Firefly luciferase has become one of the more widely used reporter proteins and is an excellent tool for the study of gene expression, given that the amount of light emitted is directly proportional to luciferase activity (4).

The luciferase assay is fast and sensitive, differentiating itself from the CAT (chloramphenicol acetyltransferase) assay because it does not require a radioactive substrate.

References

1. Eun, H. (1996). Marker/Reporter enzymes. Enzymology Primer for Recombinant DNA Technology, 567-645. doi:10.1016/b978-012243740-3/50011-9

2. McNabb, D. S., Reed, R., & Marciniak, R. A. (2005). Dual luciferase assay system for rapid assessment of gene expression in Saccharomyces cerevisiae. Eukaryotic Cell, 4(9), 1539-1549. doi:10.1128/ec.4.9.1539-1549.2005

3. Fraga, H. (2008). Firefly luminescence: A historical perspective and recent developments. Photochemical & Photobiological Sciences, 7(2), 146-158. doi:10.1039/b719181b

4. Younes, A., Lukyanenko, Y. O., Lyashkov, A. E., Lakatta, E. G., & Sollott, S. J. (2011). A bioluminescence method for direct measurement of phosphodiesterase activity. Analytical Biochemistry, 417(1), 36-40. doi:10.1016/j.ab.2011.05.036

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Peptides and proteins are guaranteed for 3 months from date of receipt.