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Method of detection of transcription factor expression activity by luciferase reporter gene system

A luciferase and transcription factor technology, applied in the field of molecular biology and cell biology, can solve the problems of long time and complicated operation, and achieve the effect of low cost, broad application prospect and excellent market potential.

Inactive Publication Date: 2017-02-15
MIAOSHUN SHANGHAI BIOTECH CO LTD
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  • Abstract
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Problems solved by technology

However, the new ELISA detection method still has the technical problems of cumbersome operation and long time-consuming

Method used

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  • Method of detection of transcription factor expression activity by luciferase reporter gene system
  • Method of detection of transcription factor expression activity by luciferase reporter gene system
  • Method of detection of transcription factor expression activity by luciferase reporter gene system

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] The main reagents and instruments purchased:

[0069] Promoter enhancer reporter vector pGL3 / 4-basic (plasmid) was purchased from Promega, USA; PBS (phosphate buffer saline) was purchased from HyClone; Lipofectamin TM 2000 liposomes were purchased from Invitrogen; the fluorescence expression intensity detector was Promega Glomax 20 / 20 bio / chemiluminescence detector. Reagent preparation:

[0070] 100ml cell lysis buffer A (pre-prepared before the experiment), in which:

[0071]

[0072] 6988μl luciferase activity detection buffer B (take 2*24 wells as an example, prepared now after the start of the experiment), of which:

[0073]

[0074] To start the experiment, the operation steps are as follows:

[0075] (1): Defining the protein composition of the transcription factor, determining the target gene promoter sequence that can bind to the transcription factor, using genomic DNA as a template, amplifying by PCR reaction to obtain the target gene promoter regulato...

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Abstract

The invention provides a method of detection of transcription factor expression activity by a luciferase reporter gene system; the method comprises the steps: based on the luciferase characteristics of being lasting, stable and easy to detect, co-transfecting cells with a luciferase reporter gene vector containing a target gene promoter and a transcription factor expression plasmid; after the cells are cultured, carrying out lysis of a certain number of cells with a cell lysis buffer liquid, collecting a lysate containing luciferase, and centrifuging to take a supernatant; and then real-timely detecting luciferase expression intensity data in a luciferase activity detection buffer liquid, at the same time, detecting the luciferase concentration, correcting, and then calculating to obtain the transcription factor expression quantity, namely the transcription factor expression activity needing to detection. The method is simpler and more feasible in whole operation process, shorter in time consuming, higher in flux, relatively high in repeatability and accuracy, and lower in operating costs, thereby having broad application prospects, and having excellent market potential.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and cell biology, and in particular relates to a method for detecting expression activity of transcription factors by a luciferase reporter gene system. Background technique [0002] Nucleic acid sequences that regulate gene expression on the same nucleic acid chain are called cis-acting elements, including promoters, enhancers, attenuators, etc.; transcription factors are also called trans-acting factors, which are a specific action on The cis-acting element in the promoter region is a protein molecule that can regulate the expression of genes on different nucleic acid chains. For a transcription factor to function, it must have a certain activity, which depends on the number of transcription factors and their binding affinity to cis-acting elements. It can be seen that detecting the activity of transcription factors is of great significance for elucidating the regulation of the expre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/66G01N33/68
CPCC12Q1/66G01N33/68G01N2333/47G01N2333/90241
Inventor 张金保陈燕
Owner MIAOSHUN SHANGHAI BIOTECH CO LTD
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