Broadbean root system proteomics analysis sample preparation method

A proteomics and sample preparation technology, which is applied in the field of sample preparation for faba bean root proteomics analysis, can solve the problems of late start of protein research and no substantial work progress, and achieves low experimental operation cost, easy operation, and repeatability. good effect

Active Publication Date: 2017-06-30
QINGHAI UNIVERSITY
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The research on protein in broad bean started relatively late, and the main research focused on protein extraction, separation, and functional properties at the food processing level, while there was only a theoretical review of proteomics, but no substantial work progress

Method used

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  • Broadbean root system proteomics analysis sample preparation method
  • Broadbean root system proteomics analysis sample preparation method
  • Broadbean root system proteomics analysis sample preparation method

Examples

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Effect test

Embodiment 1

[0048] Sample preparation method for proteomic analysis of faba bean roots

[0049] The sample preparation method of broad bean root proteomics analysis disclosed in the application comprises the following steps:

[0050] Weigh broad bean root system → liquid nitrogen grinding → phenol extract → Tris equilibrium phenol saturated solution → centrifuge to collect the upper layer of phenol → 0.1mol / L ammonium acetate-methanol solution → centrifuge to collect the precipitate → wash with methanol → centrifuge to collect the precipitate → vacuum dry the precipitate → Add sample lysate→centrifuge to take supernatant→protein quantification→subpackage and freeze→sample hydration→sample preparation is complete

[0051] (1) Weighing fresh samples: Accurately weigh 1 g of fresh samples of faba bean roots with an electronic balance.

[0052] (2) Grinding: The mortar was pre-cooled in a -20°C refrigerator in advance, the sample was weighed, placed in the pre-cooled mortar, quickly frozen w...

Embodiment 2

[0072] Comparison of four root protein extraction methods

[0073] 1. Trichloroacetic acid / acetone method to prepare samples for proteomic analysis of faba bean roots

[0074] Weigh 0.5g root sample into a liquid nitrogen pre-cooled mortar, add liquid nitrogen and grind to powder, transfer the powder to a 10mL centrifuge tube, add 5ml extract solution 1, vortex for 1min, and stand at -20°C for 2h or overnight. Then centrifuge at 35000r / min for 15min at 4°C and discard the supernatant. The obtained precipitate was washed with pre-cooled acetone for 3 to 5 times, and freeze-dried at -20°C for further use. Extract 1 (100ml) formula: phenylmethylsulfonyl fluoride (PMSF): 0.0174g; β-mercaptoethanol: 70μl; dilute to 100mL with 10% TCA / acetone.

[0075] 2. Tris-Hcl method to prepare samples for proteomic analysis of faba bean roots

[0076] Take 2g root sample and grind it, weigh 1g sample, add 10ml pre-cooled 0.2mol / L Tris-HCl, pH8.0, 4℃, extract for 6h, 4℃, centrifuge at 4000r / ...

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Abstract

The present invention discloses a broadbean root system proteomics analysis sample preparation method, which taking a fresh broadbean root system; grinding with liquid nitrogen; adding a phenol extraction liquid; adding a Tris balanced phenol saturated solution; carrying out centrifugation to collecting the upper layer phenol; adding 0.1 mol/L ammonium acetate-methanol solution; carrying out centrifugation to collecting precipitate; washing with methanol; carrying out centrifugation to collecting precipitate; carrying out vacuum drying on the precipitate; adding a sample lysate; carrying out centrifugation, and taking the supernatant; carrying out protein quantitation; sub-packaging, and freezing; carrying out sample hydration; and completing the sample preparation. According to the present invention, the broadbean root system proteomics analysis sample preparation method has advantages of simple experiment steps, easy operation, good repeatability, and easy studying and easy grasping by the technical staff having the experiment background; and the selected experiment reagents are the two-dimensional electrophoresis conventional reagents, and the expensive and rare reagents are not used, such that the experimental operation cost is low.

Description

technical field [0001] The application belongs to the field of biochemistry, and in particular relates to a sample preparation method for broad bean root proteomics 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. [0003] Proteomics is the research object of all the proteins encoded by the genome in living cells. Its research is considered to be one of the core contents of life science research in the post-genome era and has become a research hotspot and has attracted much attention. [0004] Two-dimensional electrophoresis (two-dimensional electrophoresis, 2-DE) can perform qualitative or quantitative comprehensive analysis on the expression of proteins in biological cells or tissues, especially for revealing changes in protein expression under different conditions. The prepa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31G01N1/28G01N1/34
CPCG01N1/286G01N1/34G01N21/314G01N2001/2866G01N2021/3185
Inventor 李萍刘玉皎
Owner QINGHAI UNIVERSITY
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