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Method for in situ quantification of cell membrane protein expression by nanozyme

A technology for protein expression and in situ quantification, which is applied in the field of in situ quantification of cell membrane proteins using nanozymes, which can solve the problems of tediousness, interference of background impurities, and difficulty in fast and accurate quantification of cell membrane proteins.

Active Publication Date: 2019-12-20
INST OF HIGH ENERGY PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional method faces many problems, such as the inevitable protein loss in the process of membrane protein extraction and purification, direct analysis of crude protein or complex biological samples will face the interference of background impurities, the process is complicated and cumbersome, making it difficult to realize the fast and accurate quantification of

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  • Method for in situ quantification of cell membrane protein expression by nanozyme
  • Method for in situ quantification of cell membrane protein expression by nanozyme
  • Method for in situ quantification of cell membrane protein expression by nanozyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Preparation of Nanozyme by Ligand Exchange Method for Integrin GPIIb / IIIa

[0051] In this embodiment, gold nanoparticles are used, and the targeting peptide is a polypeptide targeting cell membrane protein integrin GPIIb / IIIa.

[0052] 1) Preparation of gold nanoparticles protected by sulfhydryl small molecules

[0053] Mix 600 μL of 150 mM cystamine dihydrochloride with 20 mL of 3.0 mM HAuCl at room temperature 4 Mix to dissolve and stir for 30 minutes. Then add 25 μL of NaBH at a concentration of 500 mM 4 Aqueous solution, after stirring and reacting at room temperature for 30 minutes, a red colloidal dispersion was obtained. Afterwards, the colloidal gold nanoparticles were concentrated by centrifugation at 7500 rpm for 5 minutes using an ultrafiltration tube with a molecular weight cut-off of 100 kDa.

[0054] 2) Preparation of nanozyme containing gold nanoparticle core and polypeptide shell

[0055] 1 mL contains 2.5 mg of polypeptide (SEQ ID NO.1:...

Embodiment 2

[0056] Example 2: In situ quantification of cell membrane protein integrin GPIIb / IIIa using nanozymes

[0057] Utilize the nanozyme prepared in embodiment 1 to carry out the method for quantifying the expression amount of cell membrane protein in situ by enzyme-linked immunoassay, comprising the following steps:

[0058] 1) Establish a standard curve

[0059] 2ug / mL polyclonal antibody against the target integrin GPIIb / IIIa (human platelet membrane glycoprotein GPIIb / IIIa ELISA detection kit, Beijing Jiehui Bogao Biotechnology Co., Ltd.) was first coated on the microtiter plate with 100uL / well After that, 3% BSA in PBS was added to each well plate to block non-specific binding sites for 1 hour, followed by 50 μL of 0, 31.25, 62.5, 125, 250 and 375 ng mL -1 Integrin GPIIb / IIIa (Human Platelet Membrane Glycoprotein GPIIb / IIIa ELISA Detection Kit, Beijing Jiehui Bogao Biotechnology Co., Ltd.) standard solution was added to the well plate, and then 50 μL of 250 nM concentration p...

Embodiment 3

[0065] Example 3: Preparation of nanozyme by ligand exchange method for integrin αvβ3

[0066] In this embodiment, silver nanoparticles are used, and the targeting peptide is a polypeptide targeting human cell membrane protein integrin αvβ3.

[0067] 1) Preparation of silver nanoparticles protected by thiol small molecules

[0068] Mix 500 μL of 150 mM cystamine dihydrochloride with 15 mL of 2.0 mM AgNO at room temperature 3 Mix and stir for 30 minutes. Then add 20 μL of NaBH at a concentration of 400 mM 4 Aqueous solution, after stirring and reacting at room temperature for 30 minutes, a yellow colloidal dispersion was obtained. Afterwards, the colloidal silver nanoparticles were concentrated by centrifugation at 7500 rpm for 5 minutes using an ultrafiltration tube with a cut-off molecular weight of 100 kDa.

[0069] 2) Preparation of nanozyme containing silver nanoparticle core and polypeptide shell

[0070] 1 mL contains 2.5 mg of the polypeptide (SEQ ID NO.2: H 2 N-G...

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Abstract

The invention relates to a method for carrying out in-situ quantitation on expression quantity of cell membrane protein by using nano-enzyme. The method comprises the steps of enabling the nano-enzyme to be contacted with the cell membrane protein, and carrying out the in-situ quantitation on the expression quantity of the cell membrane protein by using the nano-enzyme combined with the cell membrane protein, wherein the nano-enzyme contains targeting peptide combined with the cell membrane protein. After the method provided by the invention is adopted, the in-situ quantitation can be rapidly, accurately and visually carried out on the expression quantity of the cell membrane protein.

Description

technical field [0001] The invention relates to a method for quantifying cell membrane proteins, and more specifically, the invention relates to a method for using nanozymes to quantify cell membrane proteins in situ. Background technique [0002] Nanozymes are a class of mimic enzymes that have both the unique properties of nanomaterials and catalytic functions. In view of the fact that nanozymes not only have high catalytic activity of natural enzymes, but also have stable and economical characteristics, since 2007, the research on nanozymes has risen rapidly, and the scope of research has gradually become wider. At present, it has included materials science, physics, chemistry , biology, medicine, and the environment. [0003] The traditional quantification method of membrane protein includes lysing the cells, performing simple separation and purification, and then using enzyme-linked immunoassay method to quantitatively analyze the crude extract of the separated protein...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/62G01N21/31G01N21/64
CPCG01N21/31G01N21/6486G01N27/62
Inventor 高学云高靓
Owner INST OF HIGH ENERGY PHYSICS CHINESE ACAD OF SCI