Method for quantifying protein abundance by taking metal cluster as artificial antibody

A technology of artificial antibodies and metal clusters, applied in the detection and quantification of low-abundance proteins, can solve the problem of not being able to obtain the exact content of the target protein

Pending Publication Date: 2021-03-12
BEIJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is usually a semi-quantitative protein analysis, and cannot obtain the exact content of the target protein

Method used

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  • Method for quantifying protein abundance by taking metal cluster as artificial antibody
  • Method for quantifying protein abundance by taking metal cluster as artificial antibody
  • Method for quantifying protein abundance by taking metal cluster as artificial antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Synthetic polypeptide-protected gold clusters as artificial antibodies

[0041] In this example, the targeting peptide sequences of MMP14 are H 2 N-CHWKHLHNTKTFL-COOH (SEQ ID NO. 1), and H 2 N-HWKHLHNTKTFLC-COOH (SEQ ID NO. 2).

[0042] During the synthesis process, firstly, 5mg of polypeptide (SEQ ID NO.1) was fully dissolved in 1.45mL of ultrapure water to make an aqueous solution of polypeptide, and mixed with 0.117mL of 25mM HAuCl at room temperature 4 Stir evenly, and add 0.24 mL of NaOH solution with a concentration of 0.5 M dropwise under sufficient stirring conditions to adjust the pH value to 12. The system was kept at 37°C for 24 hours, and then centrifuged at 3700 rpm for 10 min to remove large particles with a molecular weight cut-off of 30 kDa, and the supernatant was transferred to an ultrafiltration tube with a molecular weight cut-off of 3 kDa to continue concentrating Gold clusters, so far the preparation of gold cluster 1 is completed. S...

Embodiment 2

[0043] Example 2: Quantitative detection of MMP14 abundance in protein lysates using gold cluster 1 as an artificial antibody

[0044] Using the gold cluster 1 prepared in Example 1 as an artificial antibody to quantitatively detect MMP14 in the protein lysate of the human cervical cancer cell line Caski cell mainly includes the following contents:

[0045] 1) Separation of standard protein and sample protein by polyacrylamide gel electrophoresis

[0046] Prepare two 1.5mm, 15-well 10% polyacrylamide gels. Add 3 μL of pre-stained marker and 17 μL of loading buffer solution to the loading wells at the extreme ends of each gel, and add 1, 2, 4, 6, 8, 10 pg of standard MMP14 protein in sequence next to the loading wells with markers ( MMP14 standard protein was obtained from Boster Biotechnology Company with a purity of >95%). These six sample wells are used as standard control wells, and a blank well is reserved between the sample wells and the standard wells, and only 20 μL o...

Embodiment 3

[0058] Example 3: Quantitative detection of MMP14 abundance in protein lysates using gold cluster 2 as an artificial antibody

[0059] Using the gold cluster 2 prepared in Example 1 as an artificial antibody to quantitatively detect MMP14 in the human cervical cancer cell line Hela cell protein lysate mainly includes the following contents:

[0060] 1) Separation of standard protein and sample protein by polyacrylamide gel electrophoresis

[0061] Similar to the sample addition method in Example 2. Prepare two 1.5mm, 15-well 10% polyacrylamide gels. For each gel, add 3 μL of pre-stained marker and 17 μL of 1× loading buffer solution to the loading well at the extreme end, and add 0.5, 1, 2, 3, 4, 5 pg of standard MMP14 once to the loading well adjacent to the marker Protein (MMP14 standard protein is from Boster Biotechnology Company, purity>95%). These six sample wells are used as standard control wells, and a blank well is reserved between the sample wells and the standar...

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Abstract

The invention discloses a method for quantifying protein abundance by taking a metal cluster as an artificial antibody, and relates to a method for detecting and quantifying low-abundance protein in cells, tissue extracts or serum. The method comprises the following steps: specifically identifying target protein separated by polyacrylamide gel electrophoresis in cells, tissue lysate or serum by the artificial antibody, and quantitatively detecting the abundance of the target protein by analyzing an intrinsic fluorescence signal of the artificial antibody and a chemiluminescence signal generated by a catalytic substrate of the artificial antibody. The artificial antibody is composed of a metal cluster core and a targeting peptide of a targeting target protein modified on the metal cluster core. By using the method provided by the invention, the expression quantity of the protein in the cell, tissue or serum biological sample can be quickly and accurately analyzed quantitatively.

Description

technical field [0001] The invention relates to a method for detecting and quantifying low-abundance proteins in cells, tissue extracts or serum. More specifically, the present invention relates to the use of metal clusters as artificial antibodies instead of fluorescein-labeled or horseradish peroxidase-labeled antibodies in classical western blotting techniques, thereby performing protein labeling and abundance detection. Background technique [0002] Metal clusters usually consist of a few to dozens of metal atoms at the core, and organic single molecules or biological macromolecules are used as protective groups on the outside. It has the characteristics of small size and good dispersion, especially the metal clusters protected by biomolecules usually not only have the unique physical and chemical properties of nanomaterials, but also have good biorecognition and biocompatibility. In recent years, it has attracted the attention of scholars in many fields such as materia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N21/76G01N21/64
CPCG01N33/68G01N21/76G01N21/6486
Inventor 高学云高靓李娇娇
Owner BEIJING UNIV OF TECH
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