Method for preparing pre-staining luminescent protein marker

A light-emitting protein and protein technology, applied in the fields of molecular biology and genetic engineering, can solve the problems of cumbersome, occupied swimming lanes, and cumbersome experimental operations, and achieve the effects of enhancing optical signals, increasing binding sites, and widening the indication range.

Inactive Publication Date: 2017-10-27
南京赛诺博生物科技有限责任公司
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  • Abstract
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  • Application Information

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Problems solved by technology

[0005] However, the current products have certain deficiencies. The biotinylated protein marker needs to be added with anti-biotin-HRP antibody or streptavidin-HRP, which makes the experimental operation cumbersome.
In addition, only luminescent protein ma

Method used

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  • Method for preparing pre-staining luminescent protein marker
  • Method for preparing pre-staining luminescent protein marker
  • Method for preparing pre-staining luminescent protein marker

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Embodiment 1

[0023] 1. Synthesize the full gene sequence of Staphylococcus aureus immunoglobulin G-binding protein A (proteinA) and streptococcal immunoglobulin G-binding protein G (proteinG); purchase plasmid pFUSEss-CHIg-rG*03 (InvivoGen, Catalog#pfusess -rchg) and pFUSEss-CHIg-mG2b (InvivoGen, Catalog#pfusess-mchg2b) as the PCR template of the heavy chain constant region gene (rFc) of the rabbit antibody and the heavy chain constant region gene (mFc) of the mouse antibody; self-constructed The pET28a-his-MBP vector is the MBP sequence PCR template.

[0024] 2. Construction of pET28a-his-MBP vector

[0025] According to the MBP nucleic acid sequence in pMal-C2T (GenBank: JF795283.1), the his+MBP+ polyclonal sequence (SEQ ID NO.9) was designed and synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. into pET28a-his- MBP polyclonal vector.

[0026] 3. Design different combinations of gene fragments according to the molecular weight of each band in the luminescent marker. The specifi...

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Abstract

The invention provides a method for preparing a pre-staining luminescent protein marker. The method comprises the following steps: acquiring a protein A gene, a protein G gene, an MBP (Myelin Basic Protein) gene and a heavy chain constant region gene (CH gene) of a mouse and rabbit derived antibody, and cutting and combining the genes according to sizes of different proteins in a pre-staining luminescent protein marker; expressing the proteins, purifying, and mixing proteins of different molecule weights according to a certain ratio so as to obtain a luminescent protein marker; mixing the prepared luminescent protein marker with the pre-staining luminescent protein marker according to different ratios. The pre-staining luminescent protein marker prepared by using the method is capable of recognizing primary antibodies or secondary antibodies of different sources, binding sites of proteins are increased, light signals are intensified, not only are general positions of proteins indicated in the electrophoresis process and after membrane transfer, but also precise positions of the proteins can be indicated on X-ray films, no extra pre-staining marker channels are needed, no extra antibodies are needed, and the luminescent proteins can be combined with IgG or anti-rabbit and anti-mouse secondary antibodies derived from species such as human beings, rats, mice and rabbits.

Description

technical field [0001] The invention relates to a preparation method of a pre-stained luminescent protein marker, which belongs to the fields of molecular biology and genetic engineering. Background technique [0002] Protein markers are widely used in protein electrophoresis as a standard for protein molecular weight. It is a mixture of known proteins or polypeptides with different molecular weights. Western Blot needs a ruler for tracking the detection of positive antigen signals and transmembrane efficiency, so the pre-stained protein Marker was produced. Pre-stained protein markers are formed by chemically modifying each protein (or polypeptide) in a common protein marker and covalently coupling it with a blue or other colored dye. The appearance of pre-stained protein markers has brought great convenience to Western Blot. After transferring to the membrane, the researchers can determine the approximate position of the target band according to the size of the blue (or ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70
CPCC07K14/47C07K14/4722C07K2319/30C12N15/70
Inventor 方卫斌张海灵
Owner 南京赛诺博生物科技有限责任公司
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