Trace protein immunoblotting detection method

A detection method and Western blotting technology, which is applied in the field of protein detection, can solve the problems that Western blotting experiments cannot be carried out and hinder the development of scientific research, and achieve the effects of overcoming errors, avoiding loss, and improving the scope of application

Pending Publication Date: 2021-05-11
重庆生命知源科技有限公司
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  • Abstract
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  • Application Information

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Problems solved by technology

In the actual experimental process, many experiments can collect enough cells, resulting in the inability to carry out Western blot experiments, hindering the development of scientific research

Method used

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  • Trace protein immunoblotting detection method
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Embodiment 1

[0021] Hematopoietic stem cells (HSC) and progenitor cells (HP) were sorted by flow cytometry, and the sorted HSC and HP were transferred into EP tubes respectively. The number of cells in each EP tube was 20,000. Centrifuge for 5min. Remove part of the liquid so that the ratio of the number of cells to the residual liquid is 500:1ul, and record the volume of the residual liquid. Avoid disturbing the cell pellet at the bottom during removal of the liquid. Add 2× loading buffer, 1000× protease and 1000× phosphatase inhibitor mixture at a volume ratio of 1000:1:1 to lyse the cells to obtain a cell lysate. Add an equal volume of 2×loading buffer to the cell lysate, mix well and boil for 5 minutes to obtain a protein solution for loading. Configure SDS-PAGE electrophoresis gel, including 5% stacking gel and 10% separating gel. After pouring the gel into the gel plate, insert a hole-making comb with a tooth width of 1.5mm, and use a 3mm conventional gel-making comb as a control. ...

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Abstract

The invention particularly relates to a trace protein immunoblotting detection method, and belongs to the field of protein detection. The method is obtained by improving a traditional protein immunoblotting detection method, cells are directly lysed through a sample loading buffer solution, protein loss in the process that the cells are lysed through a lysate traditionally is avoided, the pore diameter of a gel making comb is adjusted to be 1.0-1.5 mm, the detection signal is enhanced, the defect that the total amount of protein donor cells required by the existing western blot technology is more than 10<6> is overcome, the method can be used for detecting the expression of a small amount of cell proteins, and the detection range of western blot is effectively expanded.

Description

technical field [0001] The invention belongs to the field of protein detection, and in particular relates to a trace protein immunoblotting detection method. Background technique [0002] Western blotting (Western blotting test), also known as Western Blot, is an experimental method commonly used in molecular biology, biochemistry and immunogenetics. The basic principle is to stain the cells or biological tissue samples treated by gel electrophoresis through specific antibodies. Information about the expression of specific proteins in the analyzed cells or tissues can be obtained by analyzing the location and depth of staining. Western blotting uses polyacrylamide gel electrophoresis, the detected object is protein, the "probe" is an antibody, and the "color development" is labeled with a secondary antibody. Protein samples separated by PAGE (polyacrylamide gel electrophoresis) are transferred to a solid phase support (such as nitrocellulose membrane). The solid phase supp...

Claims

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Application Information

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IPC IPC(8): G01N33/531G01N33/53G01N27/447
CPCG01N33/531G01N33/53G01N27/447Y02A50/30
Inventor 谢彪
Owner 重庆生命知源科技有限公司
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