Method for integrally marking drosophila proteomes by using stable isotope labeling with amino acids in cell culture (SILAC) and special culture medium

A technology of proteome and culture medium, applied in application, animal feed, animal feed, etc., can solve the problems affecting the accuracy of quantification and the sensitivity of detection

Active Publication Date: 2014-04-02
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Sury et al. used SILAC technology to label the whole fruit fly, but due to technical limitations, the labeling efficiency can only reach about 96%, which affects the quantitative accuracy and detection sensitivity.

Method used

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  • Method for integrally marking drosophila proteomes by using stable isotope labeling with amino acids in cell culture (SILAC) and special culture medium
  • Method for integrally marking drosophila proteomes by using stable isotope labeling with amino acids in cell culture (SILAC) and special culture medium
  • Method for integrally marking drosophila proteomes by using stable isotope labeling with amino acids in cell culture (SILAC) and special culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1, the preparation of SILAC marked yeast cell

[0027] 1. Preparation of special medium for SILAC labeled yeast

[0028] The components of each 1L of SILAC-labeled yeast special medium (SC medium containing heavy stable isotope-labeled amino acids) are as follows: 7g Difco yeast nitrogen source base (BD company in the United States, product number: 239210), 20g glucose (Sinopharm Chemical Reagent Co., Ltd., catalog number: 10010592), 164.3 μmol of heavy stable isotope-labeled lysine L-lysine- 13 C 6 (CIL Company of the United States, article number: CNLM-291-0.25), adenosine 20mg (Amresco Company of the United States, article number: 0325-50G0325-50G), uracil 20mg (Amresco Company of the United States, article number: 0847-250G0847-250G), 20mg Tryptophan (American Amresco Company, article number: E800-25G), 20mg histidine (American Amresco Company, article number: E806-100G), 20mg arginine (American Amresco Company, article number: 0877-100G), 20mg methioni...

Embodiment 2

[0044] Embodiment 2, the preparation of SILAC marked fruit fly special medium

[0045] 1. Preparation of special medium for SILAC labeled Drosophila

[0046] The components of each 1L of Drosophila culture medium are as follows: 0.8% by mass of Bio-Rad low-melting point agarose (Bio-Rad Hercules, CA), 10% by mass of glucose, 10% by mass of Mix 0.002% methyl p-hydroxybenzoate, 0.8% ethyl acetate and water at a final concentration, and make up to 1 liter with water.

[0047] The SILAC-labeled Drosophila-specific medium was prepared as follows: the above-mentioned Drosophila medium was coated with the SILAC-labeled yeast cell (yeast cell labeled with a heavy stable isotope) obtained from 2-1 of Example 1 to obtain a labeled SILAC labeling medium for Drosophila.

[0048] 2. Control Drosophila SILAC Light Labeling Medium

[0049] Control Drosophila SILAC light-labeled medium: the preparation method is basically the same as the SILAC-labeled Drosophila-specific medium, except tha...

Embodiment 3

[0051] Example 3, Application of SILAC Marking Drosophila Special Medium in Completely Marking Drosophila Proteome

[0052] 1. SILAC mark

[0053] 1. Materials

[0054] The wild-type Drosophila w118 strain (hereinafter referred to as Drosophila W118D; melanogaster w1118) was selected from the Bloomington Drosophila Stock Center at Indiana University for experiments. Cultivate at ℃ and 70% humidity.

[0055] 2. Mark

[0056] SILAC labeling: 100 female fruit flies w118 and 100 male fruit flies w118 were mixed on the above-mentioned SILAC-labeled fruit fly special medium to lay eggs; then the eggs were then transplanted into 3M filter paper folded 3 times and containing the above In the Drosophila culture tube of SILAC-labeled Drosophila-specific medium ( figure 2 ), about 100 eggs per tube, cultured in an incubator at 25°C under 70% humidity, hatched into larvae, developed into mature fruit flies through the pupal stage, and obtained SILAC-labeled fruit flies.

[0057] Con...

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Abstract

The invention discloses a method for integrally marking drosophila proteomes by using stable isotope labeling with amino acids in cell culture (SILAC) and a special culture medium. The special culture medium for marking drosophila by using the SILAC is prepared according to the following method: a saccharomycete body marked by using the SILAC is coated on a drosophila culture medium to obtain the special culture medium for marking the drosophila by using the SILAC. An experiment proves that the culture medium for marking the drosophila by using the SILAC can be specially used for marking the drosophila proteomes by using the SILAC, can achieve rapid and high-efficient marking, and creates conditions for preparation of quantified interior labels and high-precision quantified proteome studying.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and a special culture medium for using SILAC to integrally mark the fruit fly proteome. Background technique [0002] Fruit flies (Drosophila melanogaster) are tiny insects. Since Morgan began using it to study genetics, Drosophila has gradually become a model organism for the study of genetics, physiology, development and evolutionary biology. Drosophila has many advantages such as fast growth, easy breeding and reproduction, easy maintenance of large groups, low price, rich phenotypes, and most of the signaling pathways are consistent with higher organisms, making Drosophila become the gold bug in modern biological research. . [0003] Proteins are direct performers of biological functions. Proteomics is an emerging discipline that systematically identifies and quantifies intracellular proteins and studies their biological functions. With the completion of Drosophila g...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01K67/033A23K1/18
Inventor 徐平杨大福常蕾
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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