Firefly luciferase activity determination method

A technology of luciferase and determination method, applied in the field of determination of firefly luciferase activity, can solve the problems of unfavorable high-throughput screening and use of luciferase, poor light signal intensity, short duration, etc., and achieve high throughput , long-lasting and short-lasting effects

Active Publication Date: 2015-09-30
BEIJING ZHONGKEZIXIN TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of detecting luciferase in the prior art, the light signal intensity is poor and the duration is short, which is not conducive to the high-throughput screening and use of luciferase

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] (1) Prepare a mixed solution, containing 120μl Tris-HCl, 3mM MnCl 2 , 2 mM CaCl 2 , 1mM firefly fluorescein, 15mM aminoacetylglycine, 1.2mM phosphate, 6mM TCEP, 2mM DTT and 4mM NaHS;

[0032] (2) Adjust the pH value to 7.5;

[0033] (3) Pre-cool the mixture to 4°C;

[0034] (4) Take 0.35μl of luciferase-bound magnetic beads and add them to the mixture. The magnetic beads are M280 streptavidin magnetic beads, and incubate at 4°C with shaking;

[0035] (5) Add 0.4μM ATP, oscillate for 2s, and quickly place it in a micro-luminescence detector BPLC to measure the intensity of the fluorescence signal.

Embodiment 2

[0037] (1) Prepare a mixed solution containing 120μl Tris-HCl, 3mM MgCl 2 , 2 mM CaCl 2 , 100ng firefly luciferin, 11mM aminoacetylglycine, 1.5mM NaHPO 4 2 , 3mM TCEP;

[0038] (2) Adjust the pH value to 7.0;

[0039] (3) Pre-cool the mixture to 4°C;

[0040] (4) Take 0.4μl of magnetic beads bound with luciferase and add them to the mixture, the magnetic beads are M280 streptavidin magnetic beads, and shake for 5 seconds;

[0041] (5) Add 0.8μM ATP, oscillate for 4s, and quickly place it in a micro-luminescence detector BPLC to measure the intensity of the fluorescent signal, and then measure 40nl and 4nl of luciferase-bound magnetic beads with the same method.

Embodiment 3

[0043] (1) Prepare the mixed solution, containing 120μl Tris-HCl, 1mM MgCl 2 , 3 mM CaCl 2 , 90ng firefly luciferin, 12mM aminoacetylglycine, 0.5mM KH 2 PO 4 , 1mM Na 2 HPO 4 , 1mM Na 3 PO 4 , 4mM (NH 4 ) 2 S, 1mM NaHSO 3 ;

[0044] (2) Adjust the pH value to 8.0;

[0045] (3) Pre-cool the mixture to 4°C;

[0046] (4) Take 0.45μl of magnetic beads with luciferase and add them to the mixture. The magnetic beads are M280 streptavidin magnetic beads, and incubate at 4°C for 5s with shaking;

[0047] (5) Add 0.7μM ATP, oscillate for 5s, and quickly place it in a micro-luminescence detector BPLC to measure the intensity of the fluorescence signal.

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Abstract

The invention belongs to the field of biological detection, and particularly relates to a firefly luciferase activity determination method. The method includes the following steps that mixed liquid containing ATP, Mn2+, Ca2+, fluorescein, aminolevulinic glycylglycine and phosphate is prepared; the PH value is adjusted; the mixed liquid is precooled to 4 DEG C; biotinylation luciferase is incubated in the mixed liquid; the ATP is added, the mixture is put into a detection instrument, and the intensity of a fluorescence signal is determined. By means of the determination method, high throughput screening and using of the luciferase are facilitated.

Description

technical field [0001] The invention belongs to the field of biological detection, in particular to a method for measuring firefly luciferase activity. Background technique [0002] Luciferase is a general term for enzymes that can produce bioluminescence in nature, the most representative of which is the luciferase in a firefly named Photinuspyrali. In the corresponding chemical reaction, the fluorescence is generated from the oxidation of fluorescein, and in some cases, adenosine triphosphate (ATP) is also included in the reaction system. [0003] The color of the light emitted by luciferase depends mainly on the amino acids surrounding luciferin. Under normal conditions it emits a yellow-green light. But if you change the serine in it to aspartic acid (protein number 2d1t), it will glow red. [0004] The fluorescence generation reaction is usually divided into the following two steps: [0005] 1: Fluorescein + ATP → luciferyladenylate (luciferyladenylate) + PPi [0...

Claims

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Application Information

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IPC IPC(8): C12Q1/66
Inventor 高静蔡亦梅吴超徐潇张睿王者馥王绪敏殷金龙任鲁风
Owner BEIJING ZHONGKEZIXIN TECH
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