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Microarray chip for detecting protein expression in cell stress reaction and its preparation method, kit and detection method

A microarray chip and stress response technology, applied in biological testing, material inspection products, instruments, etc., can solve the problems of different affinity of antibody antigen reaction, affect the accuracy of test results, improper sample dilution, etc., and achieve rapid screening and evaluation , low sample volume, and simple preparation method

Active Publication Date: 2017-08-29
广东南芯医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are many difficulties that need to be overcome in the development of protein chips: 1. The basis of protein chips is antigen-antibody affinity reaction. Stable situation; 2. The abundance of different proteins in clinical samples is different, and the affinity of antibody-antigen reaction is different. Improper sample dilution is prone to false negative situations; 3. There are cross-reactions between proteins, which seriously affect the detection The accuracy of the results, as the number of detection antibodies increases, the more difficult it is to overcome cross-reactions

Method used

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  • Microarray chip for detecting protein expression in cell stress reaction and its preparation method, kit and detection method
  • Microarray chip for detecting protein expression in cell stress reaction and its preparation method, kit and detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Example 1. Preparation method of microarray chip for detecting protein expression in cell stress response

[0050] (1) Preparation of aldehyde-modified glass slides

[0051] Heat 800mL of deionized water to 95°C, add about 5g of washing powder, soak the new slide in washing powder water for 3h; take out the slide, soak in 1mM KOH deionized water solution for 1h; take out the slide, wash with deionized water Wash 3 times, 5min each time, and dry to obtain cleaned slides;

[0052] Soak the cleaned slides in an ethanol solution containing 5% aminomethoxysilane (V / V) for 30 minutes; take out the slides and wash them with acetone for 3 times, each time for 5 minutes; then wash them with deionized water for 3 times each time After 5 minutes, blow dry with nitrogen to obtain aminated glass slides;

[0053] Soak the amination-modified slide in PBS solution containing 2.5% glutaraldehyde (V / V) for 1 h; take out the slide, wash with PBS 3 times, 5 min each time; then wash 3 tim...

Embodiment 2

[0071] Example 2, Preparation of a kit for detecting protein expression in cell stress response

[0072] A kit for detecting protein expression in cell stress response, comprising the following components: (1) microarray chip for detecting protein expression in cell stress response, (2) cell lysate, (3) tissue lysate, ( 4) diluent, (5) washing solution, (6) enzyme-labeled secondary antibody, (7) luminescent substrate; the selection and function of each component are as follows:

[0073] (1) Microarray chip for detecting protein expression in cell stress response: preferably the microarray chip of Example 1;

[0074] (2) Cell lysate: containing Loading buffer, protease inhibitors, purchased from Beijing Beyond Biotechnology Co., Ltd.;

[0075] (3) Tissue lysate, containing Loading buffer and pmsf, purchased from Beijing Biyuntian Biotechnology Co., Ltd.;

[0076] (4) Diluent: it is a protein stabilizer, and the present embodiment is a 5% BSA solution; the effect is to dilute ...

Embodiment 3

[0081] Example 3, Detection method of protein expression in cell stress response

[0082] A method for detecting protein expression in cell stress response, comprising the steps of:

[0083] (1) Use tissue lysate and / or cell lysate to lyse the sample to obtain a protein lysate, and dilute it with a diluent to an appropriate detection concentration;

[0084] (2) Add 100 μL of diluted protein lysate to each well of the microarray chip, incubate at room temperature for 4 hours, remove unreacted protein lysate samples, wash 5 times with washing solution and PBS solution, and shake dry;

[0085] (3) Add 100 μL, 1000-fold diluted horseradish peroxidase-labeled goat anti-human IgG to each well of the microarray chip, incubate at room temperature for 4 hours, remove unreacted enzyme-labeled secondary antibody solution, wash with washing solution and PBS respectively Wash 5 times and shake dry;

[0086] (4) Add 100 μL of 1000-fold diluted fluorescein 555 to each well of the microarra...

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Abstract

The invention discloses a microarray chip for detecting protein expression in a cell stress reaction and its preparation method, kit and detection method. The microarray chip comprises seven stress reaction markers, can ensure inner cell protein expression change corresponding to a cell stress reaction under different stimulation conditions and realizes fast screening and evaluation. A hydroformylation-modified glass sheet is used as a solid carrier so that specific antibody bonding is stable and activity is high. The microarray chip can effectively eliminate the false positive and false negative results of the spectral chip test. The cross-reaction test result shows that the microarray chip has high index detection sensitivity and good specificity. The preparation method has simple processes, realizes fast detection, finishes the whole process in 2d, has high sensitivity and good specificity, realizes a low production cost and has a wide industrialization prospect.

Description

technical field [0001] The invention relates to the field of chip detection, more specifically, to a microarray chip for detecting protein expression in cell stress response, a preparation method, a kit and a detection method. Background technique [0002] Cellular stress is a series of adaptive changes that occur when prokaryotic or eukaryotic cells suffer from various obvious environmental changes or encounter radiation, reactive oxygen species, etc., resulting in macromolecular damage, which eventually leads to changes in gene expression, enhancing the ability of cells to resist damage and Survivability under adverse conditions. Currently, common cellular stress processes mainly include apoptosis, autophagy, cell proliferation, cell migration, cell stemness, and protein translation. [0003] Different cellular stress responses to different external stimuli are mainly reflected in differences in intracellular protein expression. For example, when cells are starved, in or...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N35/00
CPCG01N33/68G01N35/00029G01N2035/00158
Inventor 林金飞陈涛张召陈林
Owner 广东南芯医疗科技有限公司
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