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Membrane assisted separation of glycoprotein all N-linked carbohydrate chain and identification method thereof

A biological sample and glycoprotein technology, which is applied in the direction of analyzing materials, preparation of test samples, and material analysis by electromagnetic means, can solve the problems of low reaction efficiency and few types of sugar chains, and achieve high reaction efficiency and obvious effect , the effect of concise reaction steps

Inactive Publication Date: 2012-11-21
NORTHWEST UNIV(CN)
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Problems solved by technology

[0004] The invention provides a method for membrane-assisted separation of all N-linked sugar chains of glycoproteins in biological samples to solve the problems of low reaction efficiency and few types of sugar chains that can be identified in the prior art

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  • Membrane assisted separation of glycoprotein all N-linked carbohydrate chain and identification method thereof
  • Membrane assisted separation of glycoprotein all N-linked carbohydrate chain and identification method thereof
  • Membrane assisted separation of glycoprotein all N-linked carbohydrate chain and identification method thereof

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no. 2 example ）

[0051] The above step (2) can also adopt another solution (as the second embodiment), as follows:

[0052] Take a constant volume of protein solution 2mg (500μl each time) into a new 10KD ultrafiltration tube and add an equal volume of 16mol / L urea solution, shake and mix for 2min in a constant temperature shaking incubator at 550rpm, 14,000 in a centrifuge g Centrifuge for 15 min. Then add 200μL of 8mol / L urea solution to the ultrafiltration tube and centrifuge at 14,000g for 15min, discard the effluent in the collection tube. Add 150 μL of 10 mM DTT solution and shake and mix for 2 min in a constant temperature shaking incubator at 550 rpm, incubate at 56° C. for 45 min, and centrifuge at 14,000 g for 10 min. Add 150 μL of 20 mM IAM solution and shake and mix for 2 min in a constant temperature shaking incubator at 550 rpm, incubate in the dark for 20 min, and centrifuge at 14,000 g for 10 min. Add 150μL of urea solution to the ultrafiltration tube, centrifuge at 14,000g for ...

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Abstract

The invention provides a membrane assisted separation of glycoprotein all N-linked carbohydrate chain and an identification method thereof, and solves problems of low reaction efficiency and identification of few types of carbohydrates in prior art. According to the invention, a molecular sieve effect of a membrane of 8-12KD is employed to separate carbohydrate chain released from glycoprotein from protein; on a molecular sieve of 10KD, an N-linked carbohydrate chain of the glycoprotein is released by a PNGase F enzyme; the N-linked carbohydrate chain flows out through repeated centrifugation, but the protein is retained on the membrane, thereby realizing separation of the carbohydrate chain. The method has obvious carbohydrate chain separation effect, and avoids by-products caused by non-specific adsorption and chemical reactions; besides, the method has high reaction efficiency and concise reaction steps.

Description

Technical field [0001] The invention relates to a method for separating all N-linked sugar chains of glycoproteins in biological samples and the identification method thereof. Background technique [0002] Carbohydrates are biomolecules that are widely present in the body and play a variety of important roles in the body. Carbohydrate substances mainly exist in the form of carbohydrate complexes, such as glycolipids, glycoproteins, proteoglycans, peptidoglucans, lipopolysaccharides and so on. Among them, protein glycosylation is widely distributed on the cell surface and extracellular matrix. The sugar chain structure on it is related to many important biological functions, mainly related to regulating the conformation and stability of the protein, and controlling the half-life of the protein and even the cell. In addition, these sugar chain structures act as ligands that specifically bind to mediate targeted recognition of proteins, cell-to-cell and cell-to-extracellular matrix...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N27/64
Inventor 李铮杨刚龙马恬然王晔
Owner NORTHWEST UNIV(CN)
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