Biological detection method for small ubiquitin-like modification

A small ubiquitin-like, biological detection technology, applied in the field of small ubiquitin-like modified biological detection, can solve the problems of low sensitivity, large influence of antibody titer and specificity, and unfavorable wide application.

Active Publication Date: 2015-07-08
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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Problems solved by technology

However, Western blotting requires a series of experimental operations, many experimental steps, and a long experimental cycle. Moreover, the titer and specificity of the antibodies used have a great influence on the experimental results, and the sensitivity is not high, which is not conducive to wide application.

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  • Biological detection method for small ubiquitin-like modification
  • Biological detection method for small ubiquitin-like modification
  • Biological detection method for small ubiquitin-like modification

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Embodiment Construction

[0019] The biological detection method of small ubiquitin-like modification will be further described in detail mainly in conjunction with the accompanying drawings and specific examples below.

[0020] Such as figure 1 As shown, the biological detection method of small ubiquitin-like modification in one embodiment includes the following steps:

[0021] Step S110, constructing the fusion gene of the small ubiquitin-like modifier protein gene and the green fluorescent protein gene, which specifically includes the following steps:

[0022] First use PCR technology to amplify the small ubiquitin-like modifier protein gene; then, under the action of restriction endonucleases, through in vitro ligation reaction, the amplified small ubiquitin-like modifier protein gene is integrated into the vector containing the green fluorescent protein gene In this method, the small ubiquitin-like modifier protein gene is fused with the green fluorescent protein gene to obtain a fusion gene cont...

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Abstract

The invention relates to a biological detection method for small ubiquitin-like modification. Whether small ubiquitin-like modification occurs is detected by steps of constructing a gene fusion of a small ubiquitin-like modifier and a green fluorescent protein, performing compression of the gene fusion and a target protein in a cell, collecting the target protein through an immunosedimentation method and finally subjecting the target protein to molecule detection. According to the method, the target protein is eluted and observed after the target protein is collected by immunosedimentation, thus reducing experiment steps and shortening the experiment time. In addition, a total internal reflection fluorescence microscopy only excite fluorescence molecules in a 100 nm range on a cover glass, has high sensitivity and can detect the small ubiquitin-like modification in a single-molecule level. Accordingly, the method has characteristics of being rapid and sensitive.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a biological detection method for small ubiquitin-like modification. Background technique [0002] Small ubiquitin-like modification is a kind of protein post-translational modification widely present in cells. Abnormal small ubiquitin-like modifications can lead to the occurrence of various diseases such as neurodegenerative diseases, cancer, and cardiovascular and cerebrovascular diseases. Although small ubiquitin-like modifications regulate a series of key biological behaviors in cells, only a small proportion of intracellular target proteins undergo small ubiquitin-like modifications, usually less than 5% (see Creton, S.; Jentsch , S. Cell 2010, 143, 848-848e1). This low abundance greatly limits the detection of small ubiquitin-like modifications and the study of their biological functions. [0003] Traditionally, western blotting is mainly used to identify small ubiquitin-lik...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
CPCG01N33/542G01N33/554G01N33/6803
Inventor 张春阳杨用
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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