Method for enriching low-abundance protein in serum

A low-abundance protein and serum technology, applied in peptide preparation methods, chemical instruments and methods, animal/human peptides, etc., can solve the problems of low applicability, high cost of use, and high protein loss, and achieve low prices , the effect is good, and the effect of reducing the purification cost

Inactive Publication Date: 2009-11-11
JINAN UNIVERSITY
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Problems solved by technology

[0003] Although many methods are used in scientific research to selectively remove one or two high-abundance proteins, these methods generally have low purification rates and complicated operations.
Existing commercial products are expensive to use, and are easy to remove low-abundance proteins that interact with high-abundance proteins, so the applicability is not great. For example, MARS purification columns are expensive, and HPLC high-performance liquid phase with high cost is required Chromatographic technology, the purification process can only remove the 6 most abundant proteins, and still leave a lot of high-abundance proteins, and because the MARS purification column is purified under non-modified conditions, it can be purified in the natural conformation state. Proteins bound to albumin and other proteins will also be removed at the same time, resulting in excessive protein loss and affecting the results of the experiment

Method used

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  • Method for enriching low-abundance protein in serum
  • Method for enriching low-abundance protein in serum
  • Method for enriching low-abundance protein in serum

Examples

Experimental program
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Embodiment 1

[0021] Embodiment 1 Heparin affinity chromatography

[0022] In this example, heparin agarose is used as the filler of the purification column, and the sample is subjected to heparin affinity chromatography, and the specific steps are as follows:

[0023] 1. Sample pretreatment

[0024] Collect 10mL of normal human blood into a non-anticoagulant test tube, let it stand at room temperature for 30min, then centrifuge at 1500g for 30min, take the supernatant again at 5000g, centrifuge at 4°C for 20min, remove the precipitate and collect the supernatant, take 100μL of the supernatant, and use 60μL Dilute with purified water and 40 μL of acetonitrile, incubate at 40°C for 15 minutes, centrifuge at 14,000 g for 10 minutes to remove the precipitate, and collect the supernatant as the serum sample in this example.

[0025] 2. Affinity chromatography

[0026] The affinity chromatography in this example adopts the routine operations of those skilled in the art, and the instruments and...

Embodiment 2

[0044] Example 2 Optimization of Heparin Affinity Elution Conditions

[0045] In this example, the elution conditions of the samples of the present invention are optimized, and a series of different formulations are performed on the eluent in Example 1, and the elution effects are compared, so as to select the optimal elution conditions.

[0046] Eluent formulation:

[0047] (1) 10mmol / L Tris-HCl and 0.5mol / L NaCl, pH 7.0;

[0048] (2) 10mmol / L Tris-HCl and 1.0mol / L NaCl, pH 7.0;

[0049] (3) 10mmol / L Tris-HCl and 1.5mol / L NaCl, pH 7.0;

[0050] (4) 10mmol / L Tris-HCl and 2.0mol / L NaCl, pH 7.0.

[0051] At the same time, this embodiment also sets up a comparison group: untreated serum, heparin does not bind to components, and the eluent formula is only 1.5 mol / L NaCl.

[0052] Perform SDS-PAGE electrophoresis on the results after the above different treatments, such as Figure 4 as shown,

[0053] 1 is Marker, 2 is untreated serum, 3 is heparin non-binding fraction, 4-7 a...

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Abstract

The invention discloses a method for enriching low-abundance protein in serum. The method adopts heparin-agarose as a filler for a purification column; 1.5 mol/L NaCl is used as an eluant; a composition obtained from elution contains abundantly-enriched low-abundance protein; the content of high-abundance protein is very low; the method solves the problem of the interference of the high-abundance protein in the serum, obtains the low-abundance protein of the target class needed in the research and provides the research condition for the further in-depth study of disease diagnosis of a high-efficiency and special low-abundance protein identifier. The method selects the heparin-agarose as a purification material which has low price, simple and feasible chromatography process and good purification effect, can obtain serum protein with more varieties; and the heparin-agarose can be repeatedly utilized. The method can be used for manufacturing a merchandised albumen purification column and has large significance on applying functional proteomics to the search of a disease diagnosis marker, the research of disease pathogenesis and the development and popularization of medicaments.

Description

technical field [0001] The invention relates to the technical field of protein purification, in particular to a method for enriching low-abundance proteins in serum. Background technique [0002] The content and type of protein in human serum caused by disease is an important index for routine tests such as early diagnosis of various diseases, evaluation of disease, and judgment of prognosis. Modern biotechnology such as proteomics is used to find disease-related proteins. Specific protein changes are the hotspot of current research. However, since there are more than 10,000 kinds of proteins in serum, among them, there are as many as 22 kinds of high-abundance proteins such as albumin, globulin, and fibrinogen, accounting for about 99% of the total protein, thus covering up the Low-abundance proteins that may contain specific molecules make them difficult to detect. Therefore, effectively removing high-abundance proteins in serum and enriching low-abundance proteins in adv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K1/22
Inventor 何庆瑜雷霆
Owner JINAN UNIVERSITY
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