Method and kit for capturing nucleic acid binding protein

The technology of a kit and a protein protection agent is applied in the field of capturing nucleic acid-binding proteins, which can solve the problems of inability to effectively obtain nonpolyA-RBPs, and achieve the effects of high speed, good specificity and simple method.

Active Publication Date: 2018-08-21
GUANGZHOU RIBOBIO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing RBPs separation technology cannot achieve the purpose of effectively obtaining nonpolyA-RBPs, such as Oligo (dT) technology (Castello, A. et al. Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 149, 1393-1406 (2012)

Method used

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  • Method and kit for capturing nucleic acid binding protein
  • Method and kit for capturing nucleic acid binding protein
  • Method and kit for capturing nucleic acid binding protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Capture of RBP (RNA Binding Protein)

[0073] 1. Capturing RNA-protein complexes

[0074] 1.1EU incorporated into RNA

[0075] HeLa (obtained from ATCC) cells were cultured in a petri dish with a diameter of 100 mm, and the culture medium was 10 mL of high-glucose medium containing 10% FBS at 37°C, 5% CO 2 cultivated under conditions. When the cells were cultured to a density of 80%, EU was added at a final concentration of 0.25 mM, and the cells were co-incubated with EU for 16 hours to fully incorporate EU into the newly synthesized RNA in the cells.

[0076] 1.2 Washing

[0077] Cells were washed twice with PBS buffer to remove residual medium.

[0078] 1.3 Photocrosslinking

[0079] Remove PBS, place the culture dish containing the cells of step (2) on ice, 254nm (0.4J / CM 2 ) UV light for 1 min to form a covalent bond between RBP and interacting RNA.

[0080] 1.4 Fixed cells

[0081] Remove the culture dish from the ultraviolet light, add 5 mL of ...

Embodiment 2

[0143] Embodiment 2: the dose optimization of protein protection agent

[0144] Detection of RBP by silver staining method: see the test results Figure 14 . (see Table 3 for the setting of test conditions, and refer to Example 1 for other steps.)

[0145] Table 3 Conditions of protein protectant

[0146] protein protectant

[0147] Such as Figure 14 As shown, when THPTA is not added to the click reaction solution (condition 1), the protein bands can be clearly seen in a diffuse form in the silver staining test, and no clear bands can be seen, which proves that the protein is in the click reaction process. Destroyed; when adding THPTA (conditions 2, 3, 4), clear bands can be seen in the silver staining test, and the effect is more obvious as the concentration increases. When no aminoguanidine was added (condition 8), the protein condition also showed a diffuse and fuzzy shape; when the protective agent aminoguanidine was added (conditions 5, 6, 7), it could show...

Embodiment 3

[0148] Embodiment 3: optimization of lysate

[0149] Under different conditions of the lysate (Table 4), other components are the same as in Example 1, and the relative concentrations of the obtained proteins are compared (the comparison between the proteins is not the absolute concentration of itself, that is, no standard is used as the Standard curve for absolute quantification), other steps refer to Example 1. The test results show that the lysate of the present application can enrich RBP more efficiently.

[0150] Table 4 lysate conditions

[0151] Lysate Components

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Abstract

The present invention relates to a method for capturing RBP, and a corresponding kit. The method comprises: adding a nucleoside analog A to a biological sample system capable of synthesizing RNA; carrying out photoactivation treatment to generate photocrosslink between the RNA and proteins; adding a cell lysis liquid and a first molecule B; obtaining the content of the biological sample system; making the content contact a carrier D carrying a labeling molecule C; and separating the carrier to obtain the RNA-RBP complex. According to the present invention, the kit can capture RNA and RBP thereof in a broad-spectrum manner, and can solve the limitation of the incapability of capturing nonpolyA-RBP in the prior art; and with the method and the kit, RBP can be efficiently enriched, the specificity is good, and the non-RBP protein cannot be captured.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method and kit for capturing nucleic acid binding proteins. Background technique [0002] nonpolyA-RNA (including circRNAs, ppRNAs, eRNAs, tsRNAs, etc.) is related to various physiological and biochemical processes, such as transcriptional regulation and mRNA translation. nonpolyA-RNA usually functions in the form of protein complexes with RNA-binding proteins (RNA-binding proteins, RBPs), which are involved in RNA splicing, polyadenylation, sequence editing, RNA transport, maintenance of RNA stability and degradation, cell Aspects of RNA metabolism including internal localization and translational regulation. In order to study the interaction omics of nonpolyA-RNA and its RBPs, the separation and identification of nonpolyA-RNA and its RBPs are indispensable research methods, and technical means that can effectively separate nonpolyA-RNA and its RBPs are needed. Howev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/561G01N33/532
CPCG01N33/532G01N33/561G01N33/68
Inventor 张必良米格尔·埃斯特班鲍习琛郭亨彭尹梦回王玮克雷格·梅洛
Owner GUANGZHOU RIBOBIO
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