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Gaussia Assays

Gaussia-Juice Luciferase Assay

PJK´s measurement buffer Gaussia-Juice has been developed and optimized for the measurement of Gaussia luciferase (GLuc) activity.

 

Gaussia Glow-Juice Luciferase Assay

PJK´s measurement buffer Gaussia-Juice has been developed and optimized for the measurement of Gaussia luciferase activity and shows a stable light signal.

Substrates for Assays

Coelenterazine

Schema of conversion of Coelenterazine (CTZ) to Coelenteramide by Renilla and Gaussia luciferase in the presence of oxygen. For reaction no additional co factors are needed.

Coelenterazine and Benzyl-Coelenterazine are the obligate luciferin substrates for all Renilla, Gaussia, Ooplophorus, and Metridia Luciferase species. It is also the substrate for the calcium activated photoproteins (page 51) derived from Atolla, Aquoria, Halisturia, Beroe and Obelia species of marine organisms. It is the most prevalent luciferin worldwide, and is only found within marine organisms. It has extremely potent anti-oxidative properties.

Coelenterazine (native-CTZ)

nativ (synth.) substrate for Renilla and Gaussia luciferase. [2-(p- hydroxybenzyl)-6-(p-hydroxyphenyl)-8-benzyl-imidazol[1,2-a]pyrazin-3-(7H)-one, C26H21N3O3, MW 423.463 g/mol, CAS [55779-48-1]. Keep as dry solid in the dark at -20°C.  Packed in brown glass ampoules, bottled under argon. 

Benzyl Colenterazine (h-CTZ)

(synth.). 2-(4-Dehydroxy)coelenterazine, C26H21N3O2, MW: 407.464 g/mol, CAS [50909-86-9], packed in brown glass ampoules, bottled under argon. Keep as dry solid in the dark  at -20°C

Coelenterzine (native-CTZ in vivo)

For animal use only in sterile injection vials, each sterile vial is supplied with a sterile diluent.

Coelenterazine for in vivo applications

General Notes:Once mixed, coelenterazine (CTZ) solutions oxidize and gradually decay to their coelenteramide oxidation product. Dry coelenterazine compounds are only sparingly soluble in aqueous solutions, and must be dissolved in alcohols, propylene glycol, or straight B-mecaptoethanol prior to making aqueous buffer solutions. Mecaptoethanol solutions may inhibit or destroy luciferase activity but are fine for Aequorin and Obelin (calcium activated photoprotein) applications.

If DMSO is used in conjunction with CTZ you must mix and use immediately, DMSO will decompose the CTZ and will not give uniform repeatable results.
General Resuspension and Storage Solution:

To 10ml of 100% Methanol or Ethanol add 200ul of 3N or 6N HCl mix well. Take 0.25-1ml of the acidified alcohol and weigh out up to 10mgs of CTZ/ml of alcohol. Usually 1mg/ml is used, and it should go into suspension quite rapidly. 10mgs/ml is about the most concentrated and a slight precipitate may form at this concentration.
Store at –20C or better –70C. While not as good as freshly mixed CTZ, this should be good for 1-3 weeks. For accurate reproducible comparative data, freshly mixed is recommended for best results. Always protect any CTZ from light.

Benzyl Coelenterazine (h-CTZ in vivo)

For animal use only in sterile injection vials, each sterile vial is supplied with a sterile diluent.

Additional informations about Coelenterazine

In Vitro usage of CTZ:

Add to PBS buffers that are close to neutral, Aqueous buffers with pH 6.6-7.2 work best at avoiding precipitates, but the buffer chosen should be used in conjunction with the enzyme system.

The optimal activity of most marine luciferases occurs in buffers that are close to Sea Water in composition (500mM NaCl pH 7.8).
If you are using multi-well plates and have to assay each well over a given amount of time, you should allow the aqueous solution of CTZ to stand at room temperature for 25-30 minutes prior to plate reading.

If freshly mixed CTZ solutions are used, the initial activity read in the first plates will be quite a bit higher then the last plates due to the auto-oxidation of CTZ in water, this will be minimal if the 25-30 minute stabilization period is observed.

In vivo usage of CTZ:

For Renilla luciferase 25-100 micrograms/mouse via IP or IV injection will give good results.

Because of the increased turnover activity of Gaussia luciferase, higher CTZ concentrations are useful. In literature, for Gaussia luciferase CTZ concentration at a dose of 4mg/kg body weight will give good results for in vivo bioluminescent imaging when injected i.v.. For a 25g mouse, 20µl of 5mg/ml CTZ is mixed with 130µl of ice cold PBS and injected i. v. immediately before imagingis performed.

In general CTZ injection solution  should be given to using ethanolic rather than methanolic mixtures to live animals, and propylene glycol suspensions of CTZ, which may take longer to dissolve, it works quite well at solubilizing CTZ and will not cause venous injury that the alcholic suspensions incur; recommended in the case or repeated studies over many days.

(Tannous BA. Gaussia luciferase reporter assay for monitoring of biological processes in culture and in vivo.Nat Protoc.2009;4(4):582-591.)