Kit for detecting albumen and application thereof

A kit and protein technology, applied in biological testing, measuring devices, material inspection products, etc., can solve the problem that secreted proteins cannot be directly detected

Inactive Publication Date: 2019-09-17
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the defect that the existing flow cytometry and immunohistochemical reactions cannot directly detect secreted proteins, the present invention provides a protein detection kit and its application, which can be used for the detection of various types of proteins. The above kits have a wide range of applications and can be flexibly combined to detect most cytokines, secreted proteins, intracellular proteins and cell surface proteins

Method used

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  • Kit for detecting albumen and application thereof
  • Kit for detecting albumen and application thereof
  • Kit for detecting albumen and application thereof

Examples

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preparation example Construction

[0057] In the present invention, the preparation method of the conjugate of the goat anti-mouse IgG and quantum dots comprises the following steps:

[0058] S1. Dissolve the quantum dots in the activation solution, then add Sulfo-NHS and EDC solution in sequence, activate for 3-8 minutes, centrifuge to discard the supernatant, and dissolve the obtained precipitate with the activation solution to obtain an activated quantum dot solution;

[0059] The activation solution is 5mM BS buffer solution with a mass concentration of 0.01% Tween 20 and a pH value of 7.4;

[0060] S2. Mix goat anti-mouse IgG with the activated quantum dot solution obtained in step S1, and react in the dark for 10-16 hours at 4°C, then add BSA with a mass concentration of 8-15%, react at 37°C for 20-40min, and add washing solution, centrifuged to remove the supernatant, the precipitate was dissolved in the washing solution, and the supernatant was removed by centrifugation again, the precipitate was dissol...

Embodiment 1

[0099] 1. Coupling of PAMAM and goat anti-rabbit IgG:

[0100] (1) Chelating the strong positive charge at the amino terminus of PAMAM G5

[0101] Dissolve 60mg of PAMAM in 2ml of DMSO, stir at room temperature until dissolved, add 0.246g of succinic anhydride, shake the shaker at 180 for 4 hours, and operate in the dark throughout the process to obtain the modified PAMAM solution.

[0102] (2) Prepare a 3500MWCO dialysis bag, boil it with 1 L of 2% sodium bicarbonate, 1 mmol / LEDTA.Na2 solution for 10 minutes, and then filter and wash it with double distilled water.

[0103] (3) Transfer the modified PAMAM solution to a 3500MWCO dialysis bag for 24 hours of dialysis, then freeze-dry the dialysate, weigh it (0.057g), and record the freeze-dried product as cPAMAM.

[0104] (4) Configure MES buffer, take 4.26g MES and dissolve in 200ml water;

[0105] Prepare MES solution of NHS, take 0.023g NHS and dissolve in 4ml of MES buffer;

[0106] Prepare the MES solution of EDC, take ...

Embodiment 2

[0154] Kit for the detection of cytokine Hsp27

[0155] Description: MCF-7 breast cancer cells are used as positive cells (corresponding results are as follows Figure 9-A , Figure 9-A It is the flow diagram of L02 cells, the left picture is a scatter diagram and the right picture is a unimodal diagram).

[0156] Take L02 human normal liver cells as negative cells (the corresponding results are as follows Figure 9-B , Figure 9-B It is the flow diagram of MCF-7 cells, the left picture is a scatter diagram and the right picture is a single peak diagram).

[0157] Antigen Hsp27 was captured with mouse anti-Hsp27 and rabbit anti-Hsp27 as primary antibodies.

[0158] Add PAMAM-conjugated goat anti-rabbit IgG, incubate at 37°C for 30 minutes, and divide the incubated product into two;

[0159] Take one part of the above-mentioned bisected product and add quantum dot-coupled goat anti-mouse IgG, incubate at 37°C for 30 minutes, and the other part will not be treated. It is us...

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Abstract

The invention provides a kit for detecting albumen and application thereof, related to the technical field of biochemical detection. The kit provided by the invention comprises 1) a conjugate of goat anti rabbit IgG and PAMAM, 2) the conjugate of goat anti mouse IgG and a quantum dot, and 3) a mouse anti A and a rabbit anti B of a to-be-detected albumen, wherein the mouse anti A and a rabbit anti B need to satisfy detecting the same antigen and having no cross reaction. The kit provided by the invention has wide range of application and can be combined flexibly, can detect most of albumens including a cell factor, a secretory albumen, an intracellular albumen and a cell surface albumen.

Description

technical field [0001] The invention relates to the technical field of biochemical detection, in particular to a kit for detecting protein and its application. Background technique [0002] Flow cytometry (Flow Cytometry, FCM) is a technique for rapid quantitative analysis and sorting of cells or other biological particles (such as microspheres, bacteria, small model organisms, etc.) arranged in a single row in a liquid flow. As a technical platform for detection by flow cytometry, modern flow cytometry was produced in the 1960s and 1970s. After nearly 40 years of development and improvement, today's flow cytometer is very mature and widely used in all aspects from basic research to clinical practice, covering cell biology, immunology, hematology, oncology , pharmacology, genetics and clinical testing, etc., play an important role in various disciplines. [0003] Combined with immunofluorescence methods, flow cytometry can identify and count cells with different surface sp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535G01N33/533G01N33/543G01N33/68
CPCG01N33/533G01N33/535G01N33/54313G01N33/68G01N33/6863
Inventor 施维刘杨黄宜兵
Owner JILIN UNIV
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