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Method for enriching sulfydryl nitroso protein in serum

A technology of nitrosylation and mercaptonitroso, applied in the field of medicine, can solve the problems of lack of clearing interfering proteins, etc., and achieve the effects of good specificity, high accuracy and simple operation.

Pending Publication Date: 2021-11-02
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Currently, there is no description of a method for enriching thio-nitrosylated proteins from serum, especially the lack of a method to remove interfering proteins before enrichment, thereby improving the specificity of enrichment

Method used

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  • Method for enriching sulfydryl nitroso protein in serum
  • Method for enriching sulfydryl nitroso protein in serum
  • Method for enriching sulfydryl nitroso protein in serum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Collect peripheral blood from fasting humans or animals with vacuum blood collection tubes that do not contain anticoagulants. Let the collected samples stand at 4°C for 6 hours. After the samples are fully coagulated, carefully draw the supernatant into a 1.5mL centrifuge tube and centrifuge at 2000g for 5min to obtain the first supernatant. Transfer the first supernatant to a new 1.5mL centrifuge tube. mL centrifuge tubes to avoid dispersing the precipitate during the aliquoting process, and aliquot the samples at 100 μL / tube and store them at -80°C to avoid repeated freezing and thawing of the samples.

Embodiment 2

[0053] (1) Put the spin column into the collection tube, add 200 μL Protein A agarose and 600 μL glycine-hydrochloric acid buffer Ⅰ to the column in turn, centrifuge at 3000 rpm for 3 minutes, remove the filtrate to wash the spin column, repeat the above operation 4 times, Add 115 μL of the first supernatant obtained in Example 1 and 230 μL of 0.1M sodium phosphate-sodium chloride buffer into a new 1.5mL centrifuge tube, mix well, add the mixture to the cleaned spin column, and place Centrifuge at 3000rpm for 3min on a shaker at 4°C for 1-2h to obtain the first filtrate and the first precipitate.

[0054] (2) Add 2200 μL of glycine-hydrochloric acid buffer II to the spin column containing the first precipitate, and centrifuge at 3000 rpm for 3 minutes to obtain the second filtrate and the second precipitate.

[0055] (3) Combine the first filtrate and the second filtrate, take 500 μL of the mixed filtrate into a new centrifuge tube after pre-cooling for 10 minutes, add 362 μL ...

Embodiment 3

[0058] (1) According to the BCA method (reference: Walker, J.M., The bicinchoninic acid (BCA) assay for protein quantitation. Methods Mol Biol, 1994. 32: p. 5-8.) Measure the step (3) of Example 2 to obtain The protein concentration of the protein suspension was adjusted to 1 mg / mL with HENS buffer.

[0059] (2) Take a new centrifuge tube, add 100 μL of the serum with a protein concentration of 1 mg / mL obtained in step (1), and then add 2 μL of 1M MMTS to it, vortex for 1 min, incubate in the dark for 30 min, and place in the tube Add 500 μL of pre-cooled acetone, stand at -20°C for 1 hour, take it out and put it in a refrigerated centrifuge, centrifuge at 1000g at 4°C for 10 minutes, remove acetone, and obtain the fourth filtrate and the fourth precipitate;

[0060] (3) Add 100 μL HENS buffer to the fourth pellet for resuspension, then add 2 μL iodo TMT (Thermo Fisher Scientific Co., Ltd.), vortex to mix, then add 4 μL 1M ascorbic acid, vortex to mix, and incubate for 2 hours...

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Abstract

The invention belongs to the technical field of medicines, and particularly relates to a method for enriching sulfydryl nitroso protein in serum. According to the method, firstly, high-abundance interference proteins (albumin and immune globulin) in a serum sample are selectively removed, and then nitrosolated proteins in serum are further enriched through a specific labeling and affinity chromatography combined method. The method disclosed by the invention is good in specificity and simple to operate, and the problems of complex operation, large impurity influence and low enrichment efficiency of the existing mercaptonitrosylated protein enrichment are solved.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a method for enriching thionitrosylated proteins in serum. Background technique [0002] Protein S-nitrosylation (SNO) is a reversible redox modification in organisms. It is generally believed that nitric oxide (Nitric Oxide, NO) modifies the cysteine ​​sulfhydryl group (-SH) at a specific position of the protein to generate a product with sulfhydryl nitroso group (-SNO). The formation process of sulfhydryl nitrosylation is still being explored, and there are currently three ways: (1) Formation of cations (nitrosoniumcation, NO + ) reacts with cysteine ​​sulfhydryl group; (2) active oxygen promotes the formation of protein sulfur free radicals (RS·), and RS· reacts with NO to directly generate sulfhydryl nitrosoprotein; (3) transnitrosylation reaction, that is, from phase O-thiol nitrosylation of proteins to obtain -SNO. Studies have shown that protein sulfhydryl ...

Claims

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Application Information

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IPC IPC(8): C07K1/30C07K1/16
CPCC07K1/30C07K1/16
Inventor 余素红王伟玲黎武林刘敏曾蓉
Owner FUZHOU UNIV
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