Sample Preparation Method for Proteome Analysis of Mouse Brain Tissue

Abstract

The present invention relates to a sample preparation method for proteome analysis of mouse brain tissue. The method comprises the following steps: (1) apical perfusion to replace blood: take a mouse to be anesthetized and perform slow apical perfusion, stop when the liver turns gray and take out the brain Tissue; ⑵Protein extraction: Grind the brain tissue into powder and add pre-cooled Tris buffer, sonicate, incubate on a shaking table, and centrifuge to obtain protein extract Ⅰ; ⑶Heat treatment to increase protein solubility: Mix protein extract Ⅰ with The heat treatment solution was mixed and treated for 5 minutes, and then centrifuged to obtain the protein extract II; (4) Precipitation and degreasing: add the protein extract II to the pre-cooled degreasing precipitation solution CUS, and undergo precipitation, centrifugation, washing, and drying to obtain purification Protein powder; (5) Protein redissolution: Add the purified protein powder into the urea protein lysate, and centrifuge to obtain the proteome sample. The invention can economically and efficiently remove the effects of blood and lipids in mouse brain tissue, and increase the solubility of protein samples.

Description

technical field [0001] The invention relates to the field of proteomics research, in particular to a sample preparation method for mouse brain tissue proteome analysis. Background technique [0002] Life science research has entered the omics era of overall life research. With the development of genomics research, researchers have found that genes, as the carrier of genetic information, can only determine the basic form of life, and the genome knowledge obtained now cannot tell us The role of genes in living organisms and how they function, and the product protein expressed by genes is the real executor of life functions. Therefore, in order to understand genes and functions in depth, it is necessary to interpret and interpret at the protein level. However, there is no strict linear relationship between gene and protein expression. The expression of protein has the particularity of time and space, and its complexity and quantity are much higher than those of genes. The comp...

AI Technical Summary

Problems solved by technology

In the process of protein sample preparation, kits for removing albumin and immunoglobulin IgG in blood can also be used, but the kits are expensive, the sample volume that can be processed is small, and protein loss will occur during processing

[0008] (2) There is no step of heating the protein sample to increase the solubility, and the protein extraction efficiency is not good

However, the temperature of the protein lysate containing urea should not exceed 37°C, otherwise the urea will be hydrolyzed into isocyanate, and the isocyanate will modify the protein through carbamylation, resulting in artificial "spot series", affecting the separation and identification of protein samples

Therefore, according to this method, the sample cannot be heat-treated to solubilize

[0010] (3) There is no special step to remove lipids in brain tissue, resulting in lipids affecting protein separation and reducing protein spots

[0011] Brain tissue contains a lot of lipids. If the lipids are not removed, the viscous lipids will block the pores of the gel during proteome analysis, preventing the entry of proteins, resulting in fewer protein spots and a darker gel background. good effect

[0012] (4) There is no step to remove salt ions, which affects the electrophoresis voltage

Lower voltage will prolong isoelectric focusing time, cause isoelectric focusing failure or reduce protein separation efficiency

Images