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Novel protein extracting reagent and method

A protein extraction and reagent technology, applied in the field of protein extraction, can solve the problems of increasing the cost of experimental materials and labor, generating garbage and harmful products, and unfavorable environmental protection, so as to ensure repeated detection dosage, easy operation, time saving and effect of monetary cost

Inactive Publication Date: 2018-11-02
CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This increase in the amount of cells will most directly increase the cost of our experimental materials and labor costs
Secondly, this situation will consume more resources, generate more garbage and harmful products, which is extremely unfavorable to environmental protection
Furthermore, due to the large amount of cells required, it is inevitable to increase the operational error when processing the cells, which will affect the experimental results.
Finally, and most importantly, for many kinds of cells, especially primary cells, rare cells, newly cultivated cells, precious cells, non-proliferating cells, etc., we cannot obtain the amount of protein required by traditional protein extraction at all during experiments, and cannot Perform Western blot

Method used

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  • Novel protein extracting reagent and method
  • Novel protein extracting reagent and method
  • Novel protein extracting reagent and method

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Experimental program
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Effect test

Embodiment 1

[0034] A protein extraction reagent, weighing Tris 14.5~15.5mg, glycine 74~75mg, SDS 205mg, bromophenol blue 10~12mg, Tris-HCI (pH6.8) 0.5~0.6ml, glycerol 0.8~1.2ml, β- Mercaptoethanol 0.5ml, add deionized water to make the volume to 10ml. It can be stored for one year at -20°C.

[0035] 1. The protein extraction reagent is used for IFN-γ stimulation of human umbilical cord mesenchymal stem cells

[0036] (1) Isolation of primary human umbilical cord mesenchymal stem cells. Cultured to the third generation of primary human umbilical cord mesenchymal stem cells.

[0037] (2) Add 1x10 to each well of the 24-well plate 5Personal umbilical cord mesenchymal stem cells are added with different concentrations of IFN-γ (a drug that can stimulate target cells to secrete related protein factors) after they adhere to the wall. The concentrations of IFN-γ are 0ng / ml, 20ng / ml, 5ng / ml0, 100ng / ml. Cellular proteins were ready to be harvested after 48 hours of culture.

[0038] (3) Remo...

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Abstract

The invention provides a protein extracting reagent. Each 10 ml of aqueous solution of the protein extracting reagent contains 14.5-15.5 mg of Tris, 74-75 mg of glycine, 202-208 mg of SDS (sodium dodecyl sulfonate), 10-12 parts of bromophenol blue, 0.5-0.6 ml of Tris-HCl, 0.8-1.2 ml of glycerol, and 0.4-0.6 ml of beta-mercaptoethanol. The problem of detecting trace cells is solved, and detection sensitivity of Western blot is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to protein extraction. Background technique [0002] Western blot (Western blot) is the most important experimental method for protein detection in the scientific research industry. As long as protein detection is involved, Western blot must be the first choice. Western blot is an experimental method commonly used in molecular biology, biochemistry and immunogenetics. The basic principle is to stain the gel electrophoresis-treated cells or biological tissue protein samples 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. It is the most commonly used and most important protein detection technique. However, before performing Western blot, the total protein of cells or biological tissues must be extracted. Western blot is an experimental technique that uses t...

Claims

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Application Information

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IPC IPC(8): C07K1/14
CPCC07K1/145
Inventor 吴梦云李娅莎赵丽杨珂谭彬徐小惠田娜
Owner CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV