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High flux screening reverse protein chip, preparation method and its detection method

A protein chip and protein technology, applied in the field of protein chips, can solve problems such as limitations, and achieve the effects of low cost, reduced dosage, and reduced dosage

Inactive Publication Date: 2006-01-11
INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the limitations of existing protein chip technology in high-throughput drug screening, the present invention provides a reverse protein chip that can simultaneously screen multiple samples for a drug target

Method used

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  • High flux screening reverse protein chip, preparation method and its detection method
  • High flux screening reverse protein chip, preparation method and its detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: High-throughput drug screening protein chip targeting membrane receptors and screening of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) antagonists using the chip

[0027] It is known that LOX-1 is a membrane receptor mainly expressed by endothelial cells, and oxidized low-density lipoprotein (oxLDL) is its natural ligand. A molecular model was established based on the interaction of LOX-1-oxLDL, and high-throughput screening of LOX-1 receptor antagonists was carried out. The specific detection process is as follows:

[0028] 1. Surface cleaning and activation of carrier slides:

[0029] Put the slides in warm water diluted with detergent at 1:10, ultrasonicate for 5 minutes, then rinse with distilled water for several times, then rinse with 100% methanol, and dry the cleaned slides in an oven. Soak the slide in the first layer of coating solution APTS (aminopropyltrimethoxysilane, aminopropyltrimethoxysilane) for 10 minutes, wash with 95%...

Embodiment 2

[0045]Example 2: High-throughput screening of protein chips targeting estrogen receptors and high-throughput drug screening of estrogen receptor antagonists using the chips.

[0046] Estrogen receptor (ER) is a kind of nuclear receptor. When the estrogen receptor alpha (ERα) ligand binding domain (LBD) or DNA binding domain (DBD) binds to its specific ligand or DNA, it causes heat Shock proteins (HSPs) are structurally altered so that they cannot bind to coactivators and repress gene transcription.

[0047] 1. Cleaning treatment and activation of the carrier surface:

[0048] With embodiment 1.

[0049] 2. Preparation of protein chips:

[0050] Spread the purified ERα (0.5mg / ml) evenly on the surface of the activated glass slide to make it evenly adsorbed on the activated glass slide. In order to avoid the inactivation of the receptor protein, this process is carried out on ice, and then dried at 4°C. Rinse with PBS for 3 times, dry and place the samples to be tested on the...

Embodiment 3

[0059] Example 3: High-throughput screening of protein chips targeting elastase and high-throughput drug screening of elastase inhibitors using the chip.

[0060] 1. Surface cleaning and activation of carrier slides:

[0061] Put the glass slide in 10% NaOH, wash with 95% ethanol, and dry. Plate with 3ml of 1% agarose solution, soak it in distilled water after solidification, change the water every 2 hours, take it out after 6 hours, incubate it in a constant temperature incubator at 37°C, and take it out after drying.

[0062] 2. Preparation of elastase chip:

[0063] Spread a layer of elastase (0.5 mg / ml) solution evenly on the surface of the above-treated glass slide, place it in the chip hybridization box, add water to moisturize it, and place it in a refrigerator at 4°C to let the elastase naturally adsorb (5 hours), after adsorption Take it out, wash it with the Tris-HCl solution of pH7.4 for 3 times, dry it, use the chip spotter to spot the sample to be tested (Instit...

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Abstract

The present invention relates to a protein chip applicable to high-flux medicine screening process, its preparation technique and detection method. The invented high-flux screening reverse protein chip is characterized by that the protein related to medicine action target point can be uniformly fixed on the solid-phase carrier surface according to thin layer form, then the samples to be screened are distributed on the protein layer according to lattice form, and the receptor protein specific ligand labeled by label selected from fluorescence, isotope and other detectable substances and solid-phase carrier surface receptor protein are combined, the diction result display background is combined ligand uniform signal, after the sample to be detected and receptor protein are specifically combined, the positive result is reverse dark point. Said invention can be used for quickly screening lots of compounds, natural products and Chinese medicine extracts.

Description

technical field [0001] The invention relates to a protein chip suitable for high-throughput screening, a preparation method thereof and a detection method using the chip. Background technique [0002] Protein chip is a biochip technology developed as a supplement to gene chip after gene chip. Protein is the executor and embodiment of life activities. During transcription, a gene may be spliced ​​in multiple forms of mRNA, and the same protein may undergo post-translational modification in multiple forms. A proteome is not a direct product of a genome, and the number of proteins in a proteome can exceed the number of genomes. Therefore, protein expression profiles can more truly reflect the functional mechanism of an organism than mRNA expression profiles. Protein microarrays offer the possibility to analyze the entire proteome at the same time. [0003] The production of existing protein chips, first of all, according to the pre-designed method, a large number of differen...

Claims

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Application Information

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IPC IPC(8): G01N33/50
Inventor 杜冠华张天泰周勇
Owner INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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