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A fluorescent chemical sensor for detecting transcription factor NF-κBP50 and its detection method

A chemical sensor and transcription factor technology, applied in the field of biological analysis, can solve problems such as complex reaction principles, and achieve the effects of reducing non-specific reactions, simple operation, and accurate and reliable experimental results.

Active Publication Date: 2019-11-05
SHANDONG NORMAL UNIV
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Problems solved by technology

Although exponential amplification (EXPAR) may provide high amplification efficiencies within minutes, it involves complex reaction principles

Method used

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  • A fluorescent chemical sensor for detecting transcription factor NF-κBP50 and its detection method
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  • A fluorescent chemical sensor for detecting transcription factor NF-κBP50 and its detection method

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Experimental program
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Effect test

Embodiment

[0067] Experimental method steps

[0068] 1. Cell Culture and Preparation of Nuclear Extracts

[0069] Hela cells (human cervical cancer cell line) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin in an incubator containing 5% carbon dioxide at 37°C. Add 20 ng / mL tumor necrosis factor (TNF-α) to stimulate Hela cells, and incubate in the incubator for 30 min. Cell extracts were extracted using a nuclear extraction kit (ActiveMotif) according to the instructions. Protein concentration was quantified by Bradford method. Cell extracts were finally frozen and stored in minus 80°C refrigerator for later use.

[0070] 2. Protein-DNA Interaction and Exonuclease III Digestion

[0071] First prepare the double-stranded probe detection, 10 micromol per liter of p50-s probe, 10 micromol per liter of p50 anti-probe and 20 micromol per liter of auxiliary probe in 10 mmol per liter of Tris-HCl, 100 mM Moles per liter of sodium chloride, p...

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Abstract

The invention discloses a fluorescent chemosensor for detecting a transcription factor NF-kappaBp50 and a detection method thereof. The fluorescent chemosensor takes 2-aminopurine (2-AP) as a fluorescent group, target-driven spontaneous isothermal amplification and exonuclease-assisted fluorescent signal amplification strategies are adopted, ultrasensitive detection of the transcription factor is realized under the condition that no extract primer and template participates in, operation is easy and rapid, and test results are accurate and reliable. Experiments verify that detection limit of the technical scheme reaches 4.04*10<-4>mg / ml and can be comparable with a real-time fluorescent quantitative PCR method. Therefore, the technical scheme of the invention has extremely high popularization and application values.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, and in particular relates to a fluorescent chemical sensor for detecting transcription factor NF-κBp50 and a detection method thereof. Background technique [0002] DNA-binding proteins play crucial roles in genome replication, gene transcription, cell division and DNA repair processes. Most of the DNA-binding proteins play the role of transcription factors, regulating cell development, differentiation and proliferation, so transcription factors have also become targets in clinical diagnosis and drug screening. Therefore, the development of ultrasensitive detection of DNA-binding proteins is not only of great help to the in-depth study of basic biochemistry, but also of great significance to the development of new therapeutic methods and strategies for human diseases. [0003] So far, in the detection methods of DNA-binding proteins, the traditional electrophoretic shift assay (EMSA)...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 张春阳唐波张艳相东雪
Owner SHANDONG NORMAL UNIV
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