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Method for producing human internal reference protein for western-blot experiment and application

A technology of reference protein and pet-28a, applied in the field of molecular biology, can solve the problems such as failure and degradation of internal reference protein antibodies, and achieve the effect of simple separation and purification of expression products, high expression amount and good versatility.

Pending Publication Date: 2022-04-12
亚科因(武汉)生物技术有限公司
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reason for the negative result of the internal reference protein may be that the protein in the sample is degraded due to improper storage, or the antibody of the internal reference protein used in the experiment is invalid.
Therefore, there is currently no way to determine what causes the western-blot test to be negative

Method used

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  • Method for producing human internal reference protein for western-blot experiment and application
  • Method for producing human internal reference protein for western-blot experiment and application
  • Method for producing human internal reference protein for western-blot experiment and application

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Experimental program
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Embodiment 1

[0039] 1. Sequence analysis of internal reference proteins in different species.

[0040] The amino acid sequence similarities of GAPDH, β-Actin, and β-Tubulin proteins from humans, mice, rats, monkeys, dogs, and chickens were compared and analyzed, and the analysis results are as follows: figure 1 shown. Among them, the species corresponding to 1 is human, the species corresponding to 2 is mouse, the species corresponding to 3 is rat, the species corresponding to 4 is monkey, the species corresponding to 5 is dog, and the species corresponding to 6 is chicken.

[0041] Through comparative analysis, it was found that the internal reference protein sequences of human and other species had high homology.

[0042] 2. Synthesis of recombinant human GAPDH, β-Actin, β-Tubulin genes

[0043] Refer to the GAPDH (ACCESSION NUMBER: NM_002046.7), β-Actin (ACCESSIONNUMBER: X00351.1), β-Tubulin coding sequence (ACCESSION NUMBER: U83668.1) published by NCBI, and submit the optimized gene ...

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Abstract

The invention provides a method for detecting whether a western-blot experiment is successful, in the experiment, human internal reference protein is set as a positive control group, and the human internal reference protein is specifically set as human GAPDH, beta-Actin or beta-Tubulin protein. According to the invention, the optimal expression conditions of the human GAPDH, the beta-Actin and the beta-Tubulin in the escherichia coli are determined through experiments, and a large amount of high-purity human GAPDH, beta-Actin and beta-Tubulin proteins are obtained through later experiments.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a preparation method for using human internal reference protein in western-blot experiments as a positive control for detection. Background technique [0002] Due to the relative stability and broad-spectrum of expression in various tissues and cells, the internal reference protein is widely used as an internal reference in the western-blot experimental system to correct errors in the experimental process and ensure the accuracy of the experimental results. It is used as a blank Control, to detect whether the western-blot experiment is normal, and can be used as a standard for semi-quantitative analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (β-Actin), β-tubulin (β-Tubulin) are the most widely used internal reference proteins. [0003] Protein expression is an important technology in the fields of industry, medicine and basic research. According t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C12N15/70C07K1/22C07K1/34C07K1/36C12N15/53C12N15/12C12R1/19
Inventor 席永强叶健
Owner 亚科因(武汉)生物技术有限公司