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Method for separating N terminal of protein by adopting nanogold-modified graphene

A graphene and nano-gold technology, applied in the biological field, can solve the problem of insufficient material removal efficiency and achieve efficient and rapid separation

Inactive Publication Date: 2016-02-10
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the forward enrichment method, the reverse-phase removal method is expected to enrich more N-termini blocked by free amino groups due to chemical modification, however, the efficiency of material removal is relatively slightly insufficient

Method used

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  • Method for separating N terminal of protein by adopting nanogold-modified graphene
  • Method for separating N terminal of protein by adopting nanogold-modified graphene
  • Method for separating N terminal of protein by adopting nanogold-modified graphene

Examples

Experimental program
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Effect test

Embodiment 1

[0028] A kind of method that nano-gold modified graphene is used for protein N-terminal separation, concrete steps are as follows:

[0029] (1) Take a certain mass of graphene and disperse it in 10mMTris-HCl, then add 4-10 weight of dopamine, and stir at room temperature for 4-12h;

[0030] (2) After centrifuging the dopamine-coated graphene (GPDA), wash it alternately with water and ethanol several times, and then dry it under vacuum for 4-16 hours;

[0031] (3) Take a certain amount of GPDA and disperse it in 50-100ml of water, add 0.25mM anhydrous chloroauric acid, after heating to 85°C, add 10mM trisodium citrate, keep heating for 1h, centrifuge, wash with water and ethanol alternately After times, dry it in vacuum for use;

[0032] (4) Dissolve human plasma albumin in 6M guanidine hydrochloride, dilute with an equal volume of 50mM triethylamine bicarbonate, 5mM dithiothreitol, react at 60°C for 45 minutes, and then add 12.5mM iodoacetamide , react with light blocking fo...

Embodiment 2

[0037] A kind of method that nano-gold modified graphene is used for protein N-terminal separation, concrete steps are as follows:

[0038] (1) Take a certain mass of graphene and disperse it in 10mMTris-HCl, then add 4-10 weight of dopamine, and stir at room temperature for 4-12h;

[0039] (2) After centrifuging the dopamine-coated graphene (GPDA), wash it alternately with water and ethanol several times, and then dry it under vacuum for 4-16 hours;

[0040] (3) Take a certain amount of GPDA and disperse it in 50-100ml of water, add 0.25mM anhydrous chloroauric acid, after heating to 85°C, add 10mM trisodium citrate, keep heating for 1h, centrifuge, wash with water and ethanol alternately After times, dry it in vacuum for use;

[0041] (4) Dissolve bovine plasma albumin in 6M guanidine hydrochloride, dilute with an equal volume of 50mM triethylamine bicarbonate, 5mM dithiothreitol, react at 60°C for 45 minutes, and then add 12.5mM iodoacetamide , react with light blocking f...

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Abstract

The invention belongs to the technical field of biology, and in particular relates to a method for separating the N terminal of protein by adopting nanogold-modified grapheme. The method comprises the following steps: firstly synthesizing the nanogold-modified grapheme (G@PDA@Au), then carrying out enzymolysis on the protein with the closed amino-group, carrying out sulfydryl derivation on the enzymatic hydrolysate, removing peptides of the non-N terminal with amino-group derivation by utilizing G@PDA@Au, and finally detecting the N terminal of the protein by utilizing the matrix-assisted laser desorption / ionization time-of-flight mass spectrometry. The method provided by the invention can conveniently and efficiently remove the peptides of the non-N terminal, so that the identification effect for the peptide fragments of the N terminal of the protein is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for separating N-terminals of proteins by using gold nanometer modified materials. Background technique [0002] The amino acid start at the N-terminus of the protein is often different from the start position in the database. During protein translation and processing, the N-terminus may involve RNA cleavage, a variety of alternative translation initiation points, signal peptide cleavage, and post-translational modifications at various chemical levels, resulting in complex N-terminal protein complexes. sex. Identification of protein N-terminal sequences is of great significance for understanding the biological functions of proteins. [0003] Currently, methods for protein N-termini are mainly based on forward enrichment and reverse removal strategies. The main principle of the forward enrichment strategy is to first introduce a specific group at the N-terminal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C07K1/14G01N27/64
Inventor 张祥民李兰婷
Owner FUDAN UNIV
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