A kind of assay method of firefly luciferase activity

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

Active Publication Date: 2016-08-17
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 luciferin, 15mM aminoacetyl glycine, 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) Add 0.35 μl of luciferase-bound magnetic beads into the mixture, the magnetic beads are M280 streptavidin magnetic beads, and incubate at 4°C with shaking;

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

Embodiment 2

[0037] (1) Prepare a mixed solution containing 120μl Tris-HCl, 3mM MgCl 2 , 2 mM CaCl 2 , 100ng firefly luciferin, 11mM aminoacetyl glycine peptide, 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) Add 0.4 μl of luciferase-bound magnetic beads into the mixture, the magnetic beads are M280 streptavidin magnetic beads, and shake for 5 seconds;

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

Embodiment 3

[0043] (1) Prepare a mixed solution containing 120μl Tris-HCl, 1mM MgCl 2 , 3 mM CaCl 2 , 90ng firefly luciferin, 12mM aminoacetyl glycine peptide, 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) Add 0.45 μl of luciferase-bound magnetic beads into the mixture, the magnetic beads are M280 streptavidin magnetic beads, and incubate at 4°C for 5 s with shaking;

[0047] (5) Add 0.7 μM ATP, oscillate for 5 s, and quickly place it in a micro-luminescence detector BPLC to measure the intensity of the fluorescent 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 [00...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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