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A method for relative quantitative analysis of broiler glutathione S-transferase gsta2

A technology for quantitative analysis of glutathione, applied in the field of biochemical analysis, can solve the problems of high cost, long monoclonal control cycle, low success rate, etc., to avoid tedious steps, improve protein detection throughput and efficiency, highly specific effect

Active Publication Date: 2019-11-15
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation of GSTA2 monoclonal antibody can improve the specificity of the antibody, reduce non-specific binding and cross-reaction, but the monoclonal control cycle is long and the cost is high; if the preparation of GSTA2 polyclonal antibody can reduce the cost, but the non-specific cross-reaction is more serious, very low success rate

Method used

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  • A method for relative quantitative analysis of broiler glutathione S-transferase gsta2
  • A method for relative quantitative analysis of broiler glutathione S-transferase gsta2
  • A method for relative quantitative analysis of broiler glutathione S-transferase gsta2

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Embodiment 1

[0036] Example 1 A method for relative quantitative analysis of broiler glutathione S-transferase GSTA2

[0037] In this embodiment, the method for relative quantitative analysis of broiler glutathione S-transferase GSTA2 comprises the following steps:

[0038] (1) GSTA2 protein extraction

[0039] Take 0.1 g of broiler liver tissue from the control group and the heat treatment group respectively (the liver samples of the heat treatment group are from broiler chickens treated at 33°C for 24 hours, and the liver samples of the control group are from broiler chickens treated at 25°C for 24 hours during the same period), according to 1: Add protein extraction buffer (50mM Tris-HCl, 0.5M NaCl, 10mM dithiothreitol, 1wt% dodecyl maltoside, pH 8.0) at a ratio of 5 (mass ratio), and extract different Treat the GSTA2 protein in the broiler tissue for 15-30 minutes, after homogenization, centrifuge at 20,000 g at 4°C for 30 minutes, then take the supernatant, and use the BCA method to ...

Embodiment 2

[0047] Example 2 A method for relative quantitative analysis of broiler glutathione S-transferase GSTA2

[0048] In this embodiment, the method for relative quantitative analysis of broiler glutathione S-transferase GSTA2 comprises the following steps:

[0049] (1) GSTA2 protein extraction

[0050] Take 0.1 g of broiler liver tissue from the control group and the heat treatment group respectively (the liver samples of the heat treatment group come from broilers treated at 33°C for 18 hours, and the liver samples of the control group come from broilers treated at 25°C for 18 hours in the same period), according to 1: Add protein extraction buffer (100mM Tris-HCl, 0.1M NaCl, 20mM dithiothreitol, 2wt% dodecyl maltoside, pH 7.2) at a ratio of 5 (mass ratio), and extract different Treat the GSTA2 protein in the broiler tissue for 15-30 minutes, after homogenization, centrifuge at 20,000 g at 4°C for 30 minutes, then take the supernatant, and use the BCA method to quantify the tota...

Embodiment 3

[0058] Example 3 A method for relative quantitative analysis of broiler glutathione S-transferase GSTA2

[0059] In this embodiment, the method for relative quantitative analysis of broiler glutathione S-transferase GSTA2 comprises the following steps:

[0060] (1) GSTA2 protein extraction

[0061] Take 0.1 g of broiler liver tissue from the control group and the heat treatment group respectively (the liver samples of the heat treatment group come from broilers treated at 33°C for 30 hours, and the liver samples of the control group come from broilers treated at 25°C for 30 hours in the same period), according to 1: Add protein extraction buffer (80mM Tris-HCl, 0.3M NaCl, 15mM dithiothreitol, 1wt% dodecyl maltoside, pH 7.5) at a ratio of 5 (mass ratio), and extract different Treat the GSTA2 protein in the broiler tissue for 15-30 minutes, after homogenization, centrifuge at 20,000 g at 4°C for 30 minutes, then take the supernatant, and use the BCA method to quantify the total...

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Abstract

The invention discloses a method for relatively quantitative analysis of a broiler glutathione S transferase GSTA2. The method comprises extracting a broiler glutathione S transferase GSTA2, carryingout digestion on the broiler glutathione S transferase GSTA2 to obtain a specific peptide fragment, detecting a son-parent ion pair of the specific peptide fragment through a liquid chromatograph / massspectrometer, acquiring the son ion signal intensity sum of the specific peptide fragment, acquiring signal intensity of the GSTA2 protein according to the son ion signal intensity sum of the specific peptide fragment, and comparing the signal intensity of GSTA2 proteins in different treated broiler samples to realize relative quantification of the GSTA2 proteins in the different treated broilersamples, wherein specific peptide fragments of the broiler glutathione S transferase GSTA2 comprise three fragments with amino acid sequences shown in the formulas of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The method has the specificity higher than that of the antibody-based Western Blot method, is free of a complex antibody preparation process and improves the chicken-derived GSTA2 proteindetection flux and efficiency.

Description

technical field [0001] The invention relates to the technical field of biochemical analysis, in particular to a method for relative quantitative analysis of broiler glutathione S transferase GSTA2. Background technique [0002] Heat stress is a widely concerned issue in broiler production. It is a common in vitro stress during the growth of animals. When the temperature of the animal's growth environment exceeds the upper limit of its metabolic stability zone, and the physical adjustment of the body cannot maintain heat balance, it will cause machine Experience the non-specific response to high temperature in the environment. A large number of studies have shown that the feed intake of broilers will drop by 14% to 17% under heat stress conditions, and it will also cause a decrease in the immunity of broilers, frequent digestive diseases, and a decrease in digestibility, which will seriously affect the survival rate and feed utilization of broilers. cause economic losses. A...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/88
CPCG01N30/88
Inventor 高杰孟庆石姬生跃夏冰张宏福唐湘方
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI