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Bioactive molecule binding target identification method based on double-head photoaffinity probe

A bioactive molecule and identification method technology, which is applied in the field of bioactive molecule binding target identification based on double-headed photoaffinity probes, can solve the problems of increasing the number of potential bioactive small molecule binding proteins, difficulty in verification, and low efficiency. , to achieve good application prospects, excellent sensitivity and accuracy, and high purity.

Pending Publication Date: 2021-04-09
THE NAVAL MEDICAL UNIV OF PLA
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  • Application Information

AI Technical Summary

Problems solved by technology

However, cells are full of hydrophobic and electrostatic interactions, and thousands of proteins with different biophysical properties and abundances, so weak or transient non-specific interactions are ubiquitous, and as the proteome With the development of science and protein spectrum identification technology, the number of potentially bioactive small molecule binding proteins identified has increased significantly, and it is difficult and inefficient to verify one by one

Method used

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  • Bioactive molecule binding target identification method based on double-head photoaffinity probe
  • Bioactive molecule binding target identification method based on double-head photoaffinity probe
  • Bioactive molecule binding target identification method based on double-head photoaffinity probe

Examples

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Embodiment 1

[0040] Example 1: Preparation, purification and characterization of double-headed photoaffinity probes

[0041] 1. Preparation of double-headed photoaffinity probes

[0042] The double-headed photoaffinity probes are composed of double-headed binding probes and double-headed capture probes in groups. Double-ended binding probes are similar in structure to double-ended capture probes, see figure 2 A, Both include the probe framework in the middle and the active molecules and biological reaction amplification systems combined on both sides of the framework.

[0043] The probe backbone of the double-head binding probe binds the same biologically active molecules on both sides, the probe backbone of the double-head capture probe binds the same photoaffinity molecules on both sides, and the probe backbone of the double-head binding probe is the same as the double-head The probe backbone of the capture probe base-pairs or specifically binds.

[0044] In this embodiment, the prob...

Embodiment 2

[0065] Embodiment 2 target identification and verification

[0066] 1. Extraction of whole-cell protein samples

[0067] (1) Cell culture and pretreatment:

[0068] Human hepatoma cell Hep3B was incubated at 37°C in 5% CO 2 In the cell culture incubator, culture in DMEM high-glucose medium containing 10% fetal bovine serum, 0.1mg / mL streptomycin, 100U / mL penicillin, and when the growth is about 80% confluent, use 0.25% trypsin to digest the cells, Add an appropriate amount of serum-containing medium to stop digestion, centrifuge at 1000g for 3min, discard the supernatant, resuspend in PBS, centrifuge at 1000g for 3min, and wash once.

[0069] (2) Protein extraction:

[0070] The above-mentioned cells were resuspended in western and IP cell lysates, placed in liquid nitrogen for 3 minutes, then placed in a water bath at 37°C for 5 minutes, repeated 3 times, centrifuged at 12000g for 5 minutes, and the supernatant was taken. Observe and compare the trypan blue staining under...

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Abstract

The invention relates to the technical field of medicines, and provides a bioactive molecule binding target identification method based on a double-head photoaffinity probe. The method comprises the following three steps: preparing the double-head photoaffinity probe, preparing a protein sample, and capturing, separating and identifying a bioactive small molecule binding target. A double-head photoaffinity probe is used for giving a whole-cell extraction protein compound, and a specific binding target is visible through ultraviolet light capture and polyacrylamide gel electrophoresis separation technologies, so the non-specificity of target identification is greatly reduced, and the method can be used for binding target identification research of bioactive small molecules, is beneficial to discovering a brand-new disease binding target, and has good application prospects.

Description

technical field [0001] The invention relates to the technical field of medicine, and relates to a method for identifying a biologically active molecule binding target, in particular to a method for identifying a biologically active molecule binding target based on a double-headed photoaffinity probe. Background technique [0002] Photoaffinity labeling technology (PAL) combines the advantages of modern molecular biology, cell biology, medicinal chemistry, analytical chemistry and other disciplines. It uses synthetic photoaffinity probe molecules to produce high activity under the irradiation of specific wavelength light. The intermediate can be directly irreversibly covalently cross-linked with the protein specifically bound by the drug molecule to achieve the capture of the drug target protein molecule. Using photoaffinity labeling technology, a large number of disease-related targets have been discovered, and the modes of interaction between various small molecules and bio...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/53
CPCG01N33/68G01N33/5306
Inventor 王冬尧汤雨潇
Owner THE NAVAL MEDICAL UNIV OF PLA
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