Stabilized formulation for luminescent detection of luciferase and nucleoside phosphates
Methods, kits and compositions containing a mixture of D-luciferin and L-luciferin for light generation with luciferase are disclosed that have improved stability when stored over time. The mixture of D-luciferin and L-luciferin can be used to detect the presence or amount of ATP or of luciferase in a sample.
1. A luciferase reaction composition comprising:
a luciferase; and
a combination of D-luciferin and L-luciferin present in the composition in a ratio from about 0.25:1 to about 4:1 of D-luciferin to L-luciferin;
wherein the composition is substantially free of adenosine triphosphate (ATP); and
wherein the composition retains at least 50% luciferase activity after being stored for at least about 1 day at a temperature from about 0° C. to about 35° C.
2. The composition of claim 1 , wherein the composition retains at least 50% luciferase activity after being stored for at least about 1 day at a temperature from about 0° C. to about 35° C. as compared to a composition that has substantially no L-luciferin.
3. The composition of claim 1 , wherein the ratio of D-luciferin to L-luciferin is from about 0.5:1 to about 2:1.
4. The composition of claim 1 , wherein the ratio of D-luciferin to L-luciferin is from about 1:1 to about 4:1.
5. The composition of claim 1 , wherein the ratio of D-luciferin to L-luciferin is about 1:1.
6. The composition of claim 1 , wherein the composition has a pH of at least about 5 and less than about 9.
7. The composition of claim 1 , wherein the luciferase is a beetle luciferase.
8. The composition of claim 1 , wherein the D-Luciferin is D-5′fluoroluciferin and wherein the L-luciferin is L-5′fluoroluciferin.
9. The composition of claim 1 , wherein the composition retains at least 50% luciferase activity after being stored for at least about 1 day at a temperature from about 20° C. to about 35° C.
10. The composition of claim 1 , wherein the composition retains at least 50% luciferase activity after being stored for at least about 1 day at a temperature from about 20° C. to about 30° C.
11. The composition of claim 1 , wherein the composition retains at least 50% luciferase activity after being stored for at least about 1 day at a temperature from about 20° C. to about 25° C.
12. The composition of claim 1 , wherein the composition retains at least 50% luciferase activity after being stored for at least about 3 days at a temperature from about 0° C. to about 35° C.
13. The composition of claim 1 , wherein the composition retains at least 50% luciferase activity after being stored for at least about 5 days at a temperature from about 0° C. to about 35° C.
14. The composition of claim 1 , wherein the composition retains at least 50% luciferase activity after being stored for at least about 10 days at a temperature from about 0° C. to about 35° C.
15. The composition of claim 1 , wherein the composition further comprises at least one additional component selected from: a buffer, protein stabilizer, magnesium ions, a metal chelator, a detergent, a defoamer, inorganic phosphate, pyrophosphate, AMP, coenzyme A, a reducing agent, or a combination thereof.
16. The composition of claim 1 , wherein the composition comprises magnesium sulfate and trans-1,2-Cyclohexanediaminetetraacetic Acid (CDTA).
17. The composition of claim 1 , wherein the composition further comprises at least one additional component selected from: 1,4-Piperazinediethanesulfonic acid, 1,2-Diaminocyclohexanetetraacetic acid, Tergitol detergent, Mazu defoamer, pyrophosphate, DTT, sulfite, thiosulfate, sodium fluoride, dodecyltrimethylammonium, hydroxypolyethoxydodecane, potassium phosphate, ethylenediaminetetraacetic acid (EDTA), or a combination thereof.
18. A kit comprising the composition of claim 1 packaged in at least one container.
19. The kit of claim 18 , wherein the composition retains at least 50% luciferase activity after being stored for at least about 1 day at a temperature from about 0° C. to about 35° C. as compared to a composition that has substantially no L-luciferin.
20. A method for determining the presence or amount of ATP in a sample comprising contacting the composition of claim 1 with a sample and detecting luminescence generated, thereby determining the presence or amount of ATP in the sample.
21. The method of claim 20 , wherein the sample comprises intact cells and/or a purified enzyme which generates or uses ATP.
22. The method of claim 20 , wherein the sample comprises a crude cell lysate or a clarified cell lysate.