Dimensional electrophoresis method suitable for proteomic analysis of broad bean root system

A technology of proteomics and two-dimensional electrophoresis, which is applied in the field of broad bean root proteomics analysis, can solve problems such as the optimization of the two-dimensional electrophoresis technology system that has not yet been seen, and achieve the effect of low operating cost, good repeatability, and simple steps

Inactive Publication Date: 2019-02-22
QINGHAI UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] There are existing literature reports on the optimization and improvement of two-dimensional electrophoresis technology for cereal crops such as wheat, rice, corn, and leguminous

Method used

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  • Dimensional electrophoresis method suitable for proteomic analysis of broad bean root system
  • Dimensional electrophoresis method suitable for proteomic analysis of broad bean root system
  • Dimensional electrophoresis method suitable for proteomic analysis of broad bean root system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 IPG strip pH range screening

[0055] Screen the three pH ranges (pH3-10, pH4-7, pH5-8) of IPG gel strips available in the market, and the two-dimensional electrophoresis diagram is as follows: figure 1 shown.

[0056] from figure 1 It can be seen from the figure that when the pH of the IPG strip is 3-10, the protein spots are mainly gathered in the range from the acidic end to the neutral end, but the acidic range is too concentrated and not well separated, and a few protein spots are closely connected to each other, or even overlap. The low-abundance protein spots around individual high-resolution protein spots are covered, making the low-abundance protein spots blurred and difficult to distinguish. When the pH of IPG strips is 5-8, the protein distribution is uniform, the distance between the protein spots is large, and the separation degree is good, but some proteins are not effectively separated, resulting in the lack of protein spot distribution. When...

Embodiment 2

[0057] Embodiment 2 Isoelectric focusing parameter setting

[0058] The setting of the first-dimension electrophoresis isoelectric focusing program directly affects the separation effect of proteins. If the focusing time is insufficient, the protein will not reach the corresponding isoelectric point. If the focusing time is too long, the pH will change, which will affect the electrophoresis results. For the focusing time in the isoelectric focusing parameters, the gradient settings of 80000v / h, 85000v / h, and 90000v / h are set, and the results are as follows figure 2 shown. Comparing the obtained two-dimensional electrophoresis patterns, it was found that the protein spots in the gel image with a focusing time of 80,000v / h did not reach the isoelectric point, causing a large number of horizontal stripes in the horizontal direction, which affected the experimental results. Focusing time 90000v / h Due to excessive focusing, electroosmosis, protein sedimentation and drift are caus...

Embodiment 3

[0059] Example 3 protein loading

[0060] There are a large number of proteins with low expression abundance in root cells, and the appropriate protein loading is very important for the two-dimensional electrophoresis process and map analysis. The gradient settings of 100ug, 200ug, 300ug, and 400ug were set for the protein loading amount, and the results are as follows image 3 As shown, it can be seen from the two-dimensional electrophoresis pattern that the loading amount of 100ug protein is too low and there are few protein spots; Excessive sample loading increases the content of salt ions and other impurities in the sample solution, resulting in the inability of the isoelectric focusing protein components to be effectively and evenly distributed on the SDS-PAGE gel, resulting in horizontal and vertical tailing; 300ug protein Sample volume, clear and smooth protein spots, good focusing effect, no horizontal stripes, no tailing, good protein separation effect, and meet the ...

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Abstract

The invention discloses a dimensional electrophoresis method suitable for proteomic analysis of a broad bean root system. The method sequentially includes the following steps with the broad bean rootsystem as a material: isoelectric focusing; strip balancing; second direction polyacrylamide gel electrophoresis; gel staining. The broad bean root system is used as the material to establish and optimize the dimensional electrophoresis method suitable for the proteomic analysis of the broad bean root system, fill the blank of the dimensional electrophoresis technology of the broad bean root system, and provide technical support for deepening the proteomics of the underground part of the broad bean. The method is simple in step, easy to operate, good in repeatability and easy to lean and graspby technicians. All reagents used are dimensional electrophoresis conventional reagents, no expensive or rare reagent is used, and the experimental operation cost is low.

Description

technical field [0001] The invention relates to a proteomic analysis method suitable for broad bean root system, in particular to a two-dimensional electrophoresis method suitable for broad bean root proteomic analysis. Background technique [0002] With the full implementation and advancement of the Human Genome Project, life science has entered the post-genome era, and its main research object has been transformed into the study of functional genomics. The function of a gene is mainly realized through its encoded protein, and the study of protein structure and function will directly elucidate the change mechanism of life under physiological and pathological conditions. Proteomics is an important omics for the study of biological functions in the post-genome era. It studies the characteristics of proteins, including protein expression levels, post-translational modifications, and protein-protein interactions. Differences in growth under normal environments can provide an o...

Claims

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Application Information

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IPC IPC(8): G01N27/447
CPCG01N27/44773
Inventor 李萍刘玉皎侯万伟
Owner QINGHAI UNIVERSITY
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