Biological chips of surface plasma resonating biological sensor, preparation and application

A surface plasmon, biosensor technology, applied in instruments, scientific instruments, measuring devices, etc., can solve problems such as insufficient weak interaction detection, low sensitivity, and inability to give quantitative information

Inactive Publication Date: 2008-11-12
SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
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Problems solved by technology

Immunoprecipitation and western blotting rely on the specificity of antibodies that bind to signaling proteins or receptors, and their sensitivity is low, which is not enough for the detection of weak interactions between proteins
Yeast two-hybrid has been used to study the interaction between insulin/insulin-like receptor (IR/IGF-1R) and its signaling protein. This method is more sensitive than immunoblotting, and the weak interaction between proteins can activate the transcription and translat

Method used

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  • Biological chips of surface plasma resonating biological sensor, preparation and application
  • Biological chips of surface plasma resonating biological sensor, preparation and application
  • Biological chips of surface plasma resonating biological sensor, preparation and application

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Embodiment

[0031] Chip preparation steps of the present invention are:

[0032] ① Immobilize the antibody on the research-grade CM5 chip according to the standard EDC / NHS chemical treatment process.

[0033] ②Cell culture:

[0034]NIH 3T3 cells were cultured conventionally, and the experimental group was NIH 3T3 cells overexpressing human recombinant IGF-1R (obtained from National Institute of Diabetes and Digestive and Kidney disease, NIH, Bethesda, MA, USA), medium: DMEM high The sugar medium also contains 10% fetal bovine serum, 100ug / ml penicillin, 100 units / ml streptomycin sulfate and 500ug / ml G-418. The culture conditions of the cells in the control group were the same as those in the experimental group except that the culture medium did not contain G-418. Both cell lines were cultured in 10 mm diameter Petri dishes in 95% air and 5% carbon dioxide at 37°C.

[0035] ③Cell lysis and harvest of IGF-1R-containing cell membrane lysate:

[0036] The composition of the cell lysate is...

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Abstract

The invention relates to a cell surface receptor biochip based on a surface plasmon resonance biosensor, a preparation method and application thereof. The biochip is characterized in that the chip is provided with a gold surface coating at a glass substrate and the gold surface is fixed with a sephadex layer which is fixed with the monoclonal antibody of the Beta subunit of the receptors of anti para-insulin and surface receptors are fixed by antibody capture. The preparation method includes that the monoclonal antibody of the Beta subunit of the receptors of anti para-insulin adopts the method of antibody capture and is fixed on the surface of CM5 chip based on the surface plasmon resonance biosensor, so as to produce the protein chip of para-insulin receptors which is applicable to the mutual action between IGF-1R and IRS-1, SHC, PI3K or GRB2 and hopeful to be applied to screening cancer-fighting drugs.

Description

technical field [0001] The invention relates to a cell surface receptor biochip based on a surface plasmon resonance biosensor, a preparation method and an application thereof. The provided chip is mainly used for studying the interaction between insulin-like tyrosine kinase receptors and intracellular upstream signaling proteins Kinetics, and effects of kinase inhibitors on interactions. It belongs to the field of protein biochip. Background technique [0002] Traditional research methods for the interaction between cell surface receptors and their downstream signaling proteins include immunoprecipitation, Western blot and yeast two-hybrid. Immunoprecipitation and western blotting rely on the specificity of antibodies that bind to signaling proteins or receptors, and their sensitivity is low, which is not enough for the detection of weak interactions between proteins. Yeast two-hybrid has been used to study the interaction between insulin / insulin-like receptor (IR / IGF-1R)...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/543G01N21/55
Inventor 曹慧敏李雪岭黄明辉赵建龙杨梦苏
Owner SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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