Determining method for phosphorylation level of calpain

A technology of phosphorylation level and calpain, which is applied in the biological field, can solve the problems of cumbersome operation process, inactivation of calpain, and great harm to human body, and achieve the effect of less muscle sample, simple operation process and less potential harm

Active Publication Date: 2016-02-17
INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Another object of the present invention is to provide a method for extracting a crude sample of calpain. The content of calpain in the protein sample extracted by the tissue lysate of the present invention is higher and the activity is higher, which solves the problem in the process of phosphorylation level determination. The problem of easy partial or total inactivation of calpain
[0005] Yet another object of the present invention is to provide a method for determining the phosphorylation level of calpain. The present invention realizes the determination of the phosphorylation level of calpain by utilizing protein immunoprecipitation and protein immune techniques, and solves the problem of determining phosphorylation levels in existing methods. When it is horizontal, it is harmful to the human body and the operation process is cumbersome.

Method used

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  • Determining method for phosphorylation level of calpain
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  • Determining method for phosphorylation level of calpain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] a) Take part of the longissimus dorsi muscle samples stored at -80°C to prepare tissue lysate, and determine the protein concentration by BCA method;

[0074] b) SDS-PAGE electrophoresis: use 8% separating gel and 4% stacking gel (Acr:Bis=37.5:1), add 50 μg protein sample to each well of the gel; the initial parameter of electrophoresis is set to 70V, when the protein strip After the band completely enters the separation gel, set the voltage to 110V and continue the electrophoresis until the bromophenol blue dye migrates to the edge of the gel bottom and ends the electrophoresis;

[0075] c) Western blotting (IB): remove the gel plate after electrophoresis, put the gel into the transfer buffer to balance; cut the PVDF membrane to the size of the gel, activate it with pure methanol for 15 seconds, and use the prepared transfer membrane exclusively Put the filter paper together in the transfer buffer to balance; use the wet transfer method, ice bath at 100V for 100 minute...

Embodiment 2

[0081] a) Take part of the longissimus dorsi muscle samples stored at -80°C to prepare tissue lysate, and determine the protein concentration by BCA method;

[0082] b) SDS-PAGE electrophoresis: use 8% separating gel and 4% stacking gel (Acr:Bis=37.5:1), add 50 μg protein sample to each well of the gel; the initial parameter of electrophoresis is set to 70V, when the protein strip After the band completely enters the separation gel, set the voltage to 110V and continue the electrophoresis until the bromophenol blue dye migrates to the edge of the gel bottom and ends the electrophoresis;

[0083] c) Western blotting (IB): remove the gel plate after electrophoresis, put the gel into the transfer buffer to balance; cut the PVDF membrane to the size of the gel, activate it with pure methanol for 15 seconds, and use the prepared transfer membrane exclusively Put the filter paper together in the transfer buffer to balance; use the wet transfer method, ice bath at 100V for 100 minute...

Embodiment 3

[0089] a) Take out the muscle sample stored at -80°C, weigh 0.5g in a frozen state, cut it into small pieces and put it into a centrifuge tube;

[0090] b) Add 6 times the volume of sarcoplasmic extract (100 mM Trisbase, 10 mM EDTA, 0.05% mercaptoethanol, pH 8.3), and homogenize on ice for 30 s (every 10 s of homogenization is separated by 20 s);

[0091] c) Centrifuge the homogenate at 10,000×g in a centrifuge at a low temperature of 4°C for 35 minutes;

[0092] d) After skimming the upper fat layer, take the supernatant and subpackage;

[0093] e) Protein quantification: BCA method, store at -80°C for later use after measuring the concentration;

[0094] f) SDS-PAGE electrophoresis: use 8% separating gel and 4% stacking gel (Acr:Bis=37.5:1), add 50 μg protein sample to each well of the gel; the initial parameter of electrophoresis is set to 70V, when the protein strip After the band completely enters the separation gel, set the voltage to 110V and continue the electrophoresi...

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Abstract

The invention discloses a determining method for the phosphorylation level of calpain. The determining method includes the steps that 1, the protein concentration of a protein sample containing calpain is adjusted to 5.83-12.5 micrograms/microliter, a phosphorylated antibody is added into the protein sample, the mass ratio of the phosphorylated antibody to the total reacting weight of protein in the protein sample containing calpain is 1 to 1750-2500, then the phophorylated antibody and the protein sample are subjected to vibration hatching at 4 DEG C to form an immune complex, and then through a protein immune precipitation method, a phosphorylated calpain sample is obtained through gathering; 2, through a protein western blot method, the phosphorylation level of the phosphorylated calpain sample obtained in the step1 is detected. Through the protein immune precipitation technology and the western blot technology, phosphorylated calpain in post-slaughter muscles can be detected, the number of needed muscle samples is small, the operation process is simple, repeatability is good, stripes obtained through detection are clear, and the test method is small in potential hurt to the human body.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a method for measuring the phosphorylation level of calpain. The technology can be directly applied to the biochemical research of sheep and other animal muscles. Background technique [0002] In the field of life science research such as medicine, the phosphorylation level of calpain in organisms is usually detected by isotope labeling method, but the method of isotope labeling detection is cumbersome and has great potential harm to the human body. However, for the research and analysis of the biochemical changes of phosphorylated calpain, as well as for the study of muscle physiology and biochemistry of other animals, the phosphorylation of calpain is essential in scientific research. Although there are relatively safe methods for measuring the phosphorylation level of proteases in the prior art, since calpain is relatively sensitive, it is easily affected by the external environment,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6887
Inventor 张德权杜曼婷陈丽李铮李欣高星李蒙高玲玲
Owner INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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