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Method for fully obtaining interstitial ribosome nascent-chain complex

A new peptide and ribosome technology, applied in the field of life sciences, can solve the problems of low extraction efficiency, difficulty in obtaining, and immobilizing ribosomes

Active Publication Date: 2015-01-07
武汉承启医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method is only suitable for extracting RNC from fresh cells
Due to the complexity of the internal environment of the tissue and the fragility of the RNC itself, it is more difficult to obtain the RNC inside the tissue sample than fresh cells
When extracting RNC from cultured cells, it is necessary to add translation elongation inhibitors (such as chloramphenicol, cycloheximide, etc.) in advance to fix the ribosomes on the ribosomes to maintain the real translation status in the cells at that time, but the tissue blocks are usually composed of multiple Composed of layers of interconnected cells, it is difficult for translation elongation inhibitors to quickly penetrate into the cells inside the tissue block and immobilize ribosomes
For cultured cells, detergents such as Triton can be used to quickly and gently break the cell membrane to release the cell contents. However, due to the tight connection between cells, it is difficult for detergents such as Triton to break the tissue block well, making the extraction efficiency very low.
Therefore, the RNC extraction process from tissue samples cannot follow the strategy for extracting RNC from cells
Acquiring complete RNC of tissue samples is a great challenge for laboratories and commercial services

Method used

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  • Method for fully obtaining interstitial ribosome nascent-chain complex
  • Method for fully obtaining interstitial ribosome nascent-chain complex
  • Method for fully obtaining interstitial ribosome nascent-chain complex

Examples

Experimental program
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Effect test

Embodiment 1

[0042] (1) Six-week-old BALB / C mice were killed by cervical dislocation, and the mice were soaked in 70% (w / v) alcohol to achieve the purpose of disinfection.

[0043] (2) Obtain mouse tissues and organs (obtain lung, liver, skeletal muscle, testis, ovary, brain, spleen, kidney, adrenal gland, heart, thymus, a total of 11 tissues), and place them in the cells containing cycloheximide (final Concentration is 0.3mg / ml) and MgCl 2 Wash twice in DPBS solution at 0-4°C and pH 7.0-7.2 (final concentration: 50 mM). For larger tissue pieces (such as liver, heart, and skeletal muscle), the estimated volume is greater than 1mm 3 ), soaked in the above solution and chopped until each piece of tissue is less than 1mm 3 , and then washed twice with the above solution.

[0044] (3) Carefully collect the tissue samples processed in step (2), blot the liquid with filter paper, put the tissue samples into the collected EP tube, and quickly put them into liquid nitrogen for storage. After q...

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Abstract

The invention discloses a method for fully obtaining an interstitial ribosome nascent-chain complex. The method comprises the following steps: cleaning fresh tissues by using an isotonic saline liquid (containing translation extension inhibitors and magnesium ions) of 0-4 DEG C; removing an excessive liquid from the cleaned tissues, and then carrying out quick freezing treatment by using liquid nitrogen until the temperature of the tissues is stable; storing the quickly frozen tissues in an environment which is not higher than -80 DEG C; carrying out storage and transportation under the condition; and extracting RNC from the stored tissue samples, so as to obtain the interstitial ribosome nascent-chain complex. According to the method, interstitial RNC long-term storage and transportation can be carried out; the RNC of the tissue samples can be effectively obtained; and the restriction of time and space is broken through, thus convenience is provided for further research of the RNC and commercializing service of the RNC.

Description

technical field [0001] The invention belongs to the technical field of life sciences, and in particular relates to a method for completely obtaining a ribosome nascent-chain complex (Ribosome Nascent-chain Complex, RNC). Background technique [0002] Translation regulation is an important regulatory level in life. In cells, some genes are translated more, some genes are translated less, and some genes are not translated at all. Therefore, it is extremely necessary to study the translation status in cells. When the cell is translating, the ribosome binds to the mRNA chain and moves, and gradually synthesizes the protein polypeptide chain (called nascent-chain) according to the triplet codon information on the mRNA. This complex is called ribosome nascent-chain complex (Ribosome Nascent-chain Complex, RNC). Only when the RNC is completely extracted can qualitative and quantitative research be carried out on the mRNA being translated. Therefore, the complete extraction of RN...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C07K1/14
CPCC07K14/47
Inventor 孙黎张弓王通金静洁
Owner 武汉承启医学科技有限公司
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