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Method for detecting activity of luciferase in sample

A technology for luciferase and sample detection, which is applied in biochemical equipment and methods, and microbial measurement/inspection, etc. It can solve the problems of unfavorable luciferase high-throughput screening and use, short duration, and poor optical signal intensity , to achieve the effect of large flux, long duration and short time

Active Publication Date: 2015-10-14
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

[0020] Add 3 mM MgCl in 200 μl Tris-HCl 2 , 4 mM CaCl 2 , 1mM luciferin, 15mM aminoacetyl glycine peptide, 1.5mM phosphate, 6mM TCEP, 2mM DTT and 4mM NaHS, adjust the pH value to 7.5, then add 100ng biotinylated luciferase, shake for 5s to generate light signal , oscillating for 2 s, and quickly placed in a Promega GloMax 96 microplate luminescence detector, adding 0.47 μM ATP to the detector to measure the intensity of the fluorescent signal, and then measuring 10 ng and 1 ng respectively in the same way.

Embodiment 2

[0022] Add 4 mM MgCl in 200 μl Tris-HCl 2 , 3 mM CaCl 2 , 1mM fluorescein, 16mM aminoacetyl glycine, 1.5mM Na 2 HPO 4 2 , 6mM (NH 4 ) 2 S and 2mM NaHS, adjust the pH value to 7.0, then add 120ng biotinylated luciferase, and incubate at 4°C to generate light signals, shake for 5s, and quickly place in the Promega GloMax 96 microplate luminescence detector, the detector Add 0.4 μM ATP, measure the intensity of the fluorescent signal, and measure 12 ng and 1.2 ng respectively in the same way.

Embodiment 3

[0024] Add 2 mM MgCl in 200 μl Tris-HCl 2 , 3 mM CaCl 2 , 0.9mM fluorescein, 14mM aminoacetyl glycine peptide, 0.5mM KH 2 PO 4 , 1mM Na 2 HPO 4 , 1mM Na 3 PO 4 , 10mM NH 4 HS, adjust the pH value to 8.0, then add 100ng biotinylated luciferase, shake for 4s to generate a light signal, shake for 3s, and quickly place it in a Promega GloMax 96 microplate luminescence detector, add 0.8μM ATP to the detector, Measure the intensity of the fluorescent signal, and then measure 10 ng and 1 ng respectively in the same way.

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Abstract

The invention relates to the field of biological detection, and in particular relates to a method for detecting activity of luciferase in a sample. The method comprises the step of incubating biotinylated luciferase in a mixed solution containing ATP, Mg<2+>, Ca<2+>, luciferin, amino acetyl glycylglycine and phosphate to generate optical signals. In the process of detecting the luciferase, the optical signal intensity is high, and the duration is long, so that the high-throughput screening and use of the luciferase are facilitated.

Description

technical field [0001] The invention belongs to the field of biological detection, in particular to a method for detecting luciferase activity in a sample. Background technique [0002] Luciferase is a general term for a class of enzymes that catalyze the oxidation and luminescence of luciferin or aliphatic aldehydes in organisms, and can be extracted from bacteria, fireflies and other organisms. The luminescent reaction catalyzed by firefly luciferase must involve the participation of ATP and firefly luciferin. [0003] Luciferase can be genetically engineered in the laboratory and used in many different experiments. The gene for luciferase can be synthesized and inserted into an organism or transfected into a cell. Researchers have used genetic engineering to make some organisms such as mice, silkworms, and potatoes synthesize luciferase. Ex vivo imaging is a powerful research tool that allows the analysis of cell populations in whole animals: different types of cells ...

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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