Molecular probe of higher plant non-cobalt-dependent methionine synthase and application of molecular probe

A technology of methionine synthase and higher plants, applied in the field of highly selective probes, can solve problems such as unclear binding of probes, and achieve the effect of high specificity and high affinity

Active Publication Date: 2020-07-31
CHANGSHU INSTITUTE OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] CN 105503879 A discloses that the structure of the probe G1-yne is similar to the compound of the present application, and it has been reported to covalently modify the tyrosine 111 residue of the Schistosoma japoni...

Method used

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  • Molecular probe of higher plant non-cobalt-dependent methionine synthase and application of molecular probe
  • Molecular probe of higher plant non-cobalt-dependent methionine synthase and application of molecular probe
  • Molecular probe of higher plant non-cobalt-dependent methionine synthase and application of molecular probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0046] Biological experiments

[0047] Protein labeling of Arabidopsis tissue crude extracts based on gel electrophoresis fluorescence scanning.

[0048] In reaction buffer (50mM HEPES, pH 7.4, 150mM NaCl, 5mM MgCl 2) to a final concentration of 2.5 μg / μL and add a final concentration of 1 μM probe G1-yne to incubate for 1 hour at room temperature. Then add SDS at a final concentration of 1% to the protein sample and perform a "click" reaction: For each reaction, add 0.2 μL of TAMRA-N to 19.2 μL of the protein sample 3 (10mM stock solution in DMSO, Lumiprobe), CuSO 4 (100 mM stock in water), THPTA (10 mM stock in water, Sigma) and sodium ascorbate (100 mM stock in water). The samples were incubated at room temperature in the dark for 1 hour, and the reaction was terminated by adding 5 μL of 5×SDS loading buffer and boiling at 95° C. for 15 minutes. Samples were applied to 10% Bis-tris denaturing gels and in-gel fluorescence scanning was performed with a FUJIFILM FLA 9000 p...

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Abstract

The invention belongs to the technical field of biology, and relates to a high-selectivity probe of non-cobalt-dependent methionine synthase in higher plants and application thereof. The highly selective probe contains sulfonyl fluoride groups and is capable of specifically and covalently labeling proteins at lysine residues. The probe can be used for detecting the activity and expression quantityof non-cobalt-dependent methionine synthase in higher plants and positioning tissues and subcells. The probe provided by the invention provides a targeted protein research tool, so that deeper research on protein expression changes and protein network maps of different plant types, different strains or different development stages can be realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a highly selective probe for cobalt-independent methionine synthase in higher plants and its application. Background technique [0002] Cobalamin-independent methionine synthase (MS), widely present in higher plants, catalyzes the transfer of methyl groups from N5-methyltetrahydrofolate to homocysteine ​​to generate methionine and tetrahydrofolate, It is directly involved in the methionine cycle, folic acid cycle and metabolism of sulfur-containing amino acids. It is closely related to DNA, protein synthesis and biological methylation, and provides intermediate products for the methylation reaction in vivo and the synthesis of polyamines and ethylene. [0003] Currently, there are no targeted antibodies and molecular probes for non-cobalt-dependent methionine synthase on the market. Therefore, there is a lack of effective antibody tools for this research field, which can be used in dif...

Claims

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Application Information

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IPC IPC(8): C09K11/06G01N21/64
CPCC09K11/06G01N21/6428G01N21/6458G01N2021/6439C09K2211/1074
Inventor 周怡青陈鑫许静远王春夏戴含章沈陈金鑫
Owner CHANGSHU INSTITUTE OF TECHNOLOGY
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