Method for detecting zebrafish M3 receptor protein based on determination of p38MAPK protein expression

A protein expression and receptor protein technology, applied in the field of physiological ecology, can solve problems such as poor binding

Active Publication Date: 2016-10-26
SHANDONG NORMAL UNIV
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Problems solved by technology

Since this method must use an antibody that specifically binds to the target protein, there is currently no antibody for the detection of the zebrafish M3 receptor, and because there are huge differences in the M3 receptor protein between tissues and species, commercially available Other M3 receptor antibodies, such as human M3 receptor antibody, mouse M3 receptor antibody, etc., have extremely poor binding to zebrafish M3 receptor, which makes western blot technique not applicable to the detection of zebrafish M3 receptor

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  • Method for detecting zebrafish M3 receptor protein based on determination of p38MAPK protein expression
  • Method for detecting zebrafish M3 receptor protein based on determination of p38MAPK protein expression

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[0031] (1) Protein sample extraction and preparation: Take zebrafish brain tissue, add cell lysate to get supernatant, transfer to a new pre-cooled microcentrifuge tube and place on ice, that is protein sample, discard the precipitate, and use Bradford protein analysis method to determine the protein concentration;

[0032] (2) SDS gel electrophoresis: prepare electrophoresis gel, carry out SDS-PAGE, the mass fraction of separation gel in described SDS gel electrophoresis is 10%, the mass fraction of stacking gel is 5%, and the processing temperature of electrophoresis process is 10 ℃ ;

[0033] (3) Membrane transfer: transfer the protein from the gel to the nitrocellulose membrane, and keep the temperature at 4°C during the transfer process;

[0034] (4) Sealing: put into 5% skimmed milk powder to seal for 1 hour, and keep the binding site without antibody on the membrane during the sealing process, so that the site is in a saturated state;

[0035] (5) Primary antibody and...

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Abstract

A method for detecting zebrafish M3 receptor protein based on determination of p38MAPK protein expression comprises the following steps: (1), preparing a zebrafish brain tissue protein sample; (2), performing SDS (sodium dodecyl sulfate) gel electrophoresis; (3), performing film transferring; (4), closing; (5), performing primary and secondary antibody inoculation; (6), performing rendering reaction; (7), calculating M3 receptor variation results. The technical defect that traditional zebrafish M3 receptor detection is limited to zero specific antibody and accordingly is unable to use western immunoblotting is overcome, the use of p38MAPK phosphorylation level to determine changes in zebrafish M3 receptor protein content is provided for the first time, and the method is high in sensitivity, good in stability and free of complex equipment; meanwhile, the method is simple and quick to perform, high in specificity and low in cost; internal acting mechanism of a cholinergic system is studied in terms of molecules, and in connection with study on zebrafish behavior changes, finally water quality can be estimated and monitored online in real time.

Description

technical field [0001] The invention belongs to the technical field of physiological ecology, and in particular relates to a method for detecting zebrafish M3 receptor protein based on western blotting technology. Background technique [0002] In recent years, with the rapid development of the economy, the problem of water pollution has become increasingly prominent, seriously affecting the balance and stability of the ecological environment. Zebrafish is a small tropical freshwater fish. In recent years, many studies have shown that zebrafish has its unique advantages in environmental toxicology research. Zebrafish has gradually become the new favorite of environmental toxicologists. Rapid monitoring of pollutants has become a research hotspot. [0003] At present, people usually measure the mortality rate, deformity rate, body length, weight, body mass index (BMI) of zebrafish, etc. However, the biological pathological damage or death of zebrafish often lags behind, and i...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/18
CPCG01N33/186G01N33/94G01N2333/70571
Inventor 任宗明张婷婷任晴李尚戈邢娜齐鲁慧子
Owner SHANDONG NORMAL UNIV
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