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Fluorescence labeling-based uridine monophosphate acidification detection method

A technology of uridine monophosphate and fluorescent labeling, which is applied in the field of biochemistry, can solve the problems that have not received attention and the modification detection method has not been established, and achieve the effect of shortening the experimental time

Pending Publication Date: 2020-10-16
INST OF BASIC MEDICINE OF SAMS
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Problems solved by technology

Among them, protein uracil mononucleotide modification (UMPylation, UMPylation) was first identified in Escherichia coli in 1975, but the substrate protein of UMPylation was limited to the glutamic acid synthase system, resulting in UMPylation has been unknown. Received attention, although it has been discovered for more than 40 years, the corresponding modification detection method has not been established

Method used

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  • Fluorescence labeling-based uridine monophosphate acidification detection method
  • Fluorescence labeling-based uridine monophosphate acidification detection method
  • Fluorescence labeling-based uridine monophosphate acidification detection method

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Embodiment Construction

[0021] The present invention will be further described according to the following examples, and the mode of the present invention includes but not limited to the following examples.

[0022] Such as figure 1 Shown, the present invention comprises the following steps:

[0023] A: Add YdiU protein and substrate to the reaction system, mix well, add 1 μl of CY3-X-UTP, and react for UMP modification at 37°C for 1 hour to obtain UMP-modified protein with CY3 label;

[0024] B. After adding the UMP-modified protein to the loading buffer, use 12% SDS-PAGE gel electrophoresis;

[0025] C Use a fluorescence imager to observe the gel under the emission of 565 nm after excitation at 553 nm.

[0026] The molecular structure of the CY3-labeled UMP donor CY3-X-UTP used in the modification reaction is as follows:

[0027]

[0028] Based on this identification method, a kit is developed, which provides UMP transferase that catalyzes uridylation of monophosphate, fluorescently labeled UT...

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Abstract

The invention discloses a fluorescence labeling-based uridine monophosphate acidification detection method. The method comprises the following steps of: adding YdiU protein and a substrate into a reaction system, mixing the substances uniformly, then adding 1 microliter of CY3-X-UTP, and carrying out UMPylation modification reaction at 30-40DEG C for 1h to obtain UMPylation modified protein with aCY3 label; after adding the UMPylation modified protein into a sample loading buffer solution, using 12% SDS-PAGE gel electrophoresis; and carrying out fluorescence imaging on the buffer solution, and carrying out imaging observation under 565nm emission after 553nm excitation. The method is used for qualitatively identifying the protein possibly subjected to uridine monophosphate modification, can determine a specific modification site by combining mass spectrometry, and can be used for screening an inhibitor of targeted uridine monophosphate transferase.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a method for detecting uridine acidification of monophosphate based on fluorescent labels. Background technique [0002] Protein is the bearer of life activities, protein post-translational modification can regulate a variety of life activities, and its imbalance can cause various diseases such as cancer. Among them, protein uracil mononucleotide modification (UMPylation, UMPylation) was first identified in Escherichia coli in 1975, but the substrate protein of UMPylation was limited to the glutamic acid synthase system, resulting in UMPylation has been unknown. It has received attention, although it has been discovered for more than 40 years, the corresponding modification detection method has not been established. With the development of protein modification omics technology, more than 40 UMP-modified proteins have been identified in recent years, indicating that UMP-modified prote...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6428G01N21/6456G01N2021/6439
Inventor 李冰清岳盈盈宋楠楠李翠玲王玮玮贾海红
Owner INST OF BASIC MEDICINE OF SAMS
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