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A method and kit for capturing nucleic acid-binding proteins

A protein protectant and protein technology, which is applied in the field of capturing nucleic acid binding proteins, can solve problems such as the inability to effectively obtain nonpolyA-RBPs

Active Publication Date: 2021-06-08
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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  • A method and kit for capturing nucleic acid-binding proteins
  • A method and kit for capturing nucleic acid-binding proteins
  • A method and kit for capturing nucleic acid-binding proteins

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 THPTA concentration Aminoguanidine Concentration Condition 1 0mM 1mM Condition 2 0.3mM 1mM Condition 3 0.9mM 1mM Condition 4 1.5mM 1mM Condition 5 0.9mM 0.5mM Condition 6 0.9mM 1mM Condition 7 0.9mM 1.5mM Condition 8 0.9mM 0mM

[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), clea...

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 Relative protein concentration (mg / ml) 150mM NaCl, 0.1% SDS, 0.5% NP40 0.873 150mM LiCl, 0.1% LiDS 0.738 500mM LiCl, 0.1% LiDS 0.789 500mM LiCl, 0.5% LiDS 0.904 500Mm LiCl, 1% LiDS 0.955

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Abstract

The present invention relates to a method for capturing RBP and a corresponding kit. The method includes adding nucleoside analog A to a biological sample system capable of synthesizing RNA; photoactivating treatment to make photocrosslinking of RNA and protein; adding cell lysis solution, adding the first molecule B; obtaining the content of the biological sample system; contacting the content with the carrier D with the labeled molecule C; separating the carrier to obtain the RNA‑RBP complex. The kit of the invention can capture RNA and its RBP more broadly, and solve the limitation that the prior art cannot obtain nonpolyA-RBP. It can efficiently enrich RBP with good specificity and will not capture non-RBP proteins.

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 Patents(China)
IPC IPC(8): C12Q1/6806
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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