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Kit for detecting protein glycosylation subtype and detection method thereof

A detection kit and a technique for isolating isoforms are applied in the field of protein and post-translational modification detection to achieve the effect of improving detection efficiency and wide applicability

Inactive Publication Date: 2015-04-08
THE FIRST AFFILIATED HOSPITAL OF MEDICAL COLLEGE OF XIAN JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problem of detection of protein glycosylation subtypes in biological samples, and provide a kit for detecting protein glycosylation subtypes and its detection method, which can not only realize the detection of a single protein glycosylation subtype semi-quantitative detection, easy to operate and low cost

Method used

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  • Kit for detecting protein glycosylation subtype and detection method thereof
  • Kit for detecting protein glycosylation subtype and detection method thereof
  • Kit for detecting protein glycosylation subtype and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1) Dissolve and dilute West African simplicifolia agglutinin (Bandeiraea simplicifolia, BS-I) with pH = 9.6 carbonate buffer to 3 μg / mL, add it to each polystyrene reaction well of a 96-well plate, and make It covered the entire bottom of the well, and 100 μL of the lectin solution was added to each well, and left overnight at 4°C.

[0036] 2) The next day, the lectin solution in the wells was discarded, and 200 μL of blocking solution was added to each well to block the parts not bound to the lectin, and incubated at room temperature for 1 h.

[0037] 3) Wash the 96-well plate 4 times with washing buffer. The washing buffer should fill up the entire reaction well each time, shake gently for 3 minutes, discard the liquid in the well and pat dry on absorbent paper.

[0038] 4) Dilute serum samples with PBS (pH=7.4) containing 0.01g / mL BSA at 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1: After 2560-fold dilution, add 100 μL to each well of a 96-well plate and incubat...

Embodiment 2

[0046] 1) Dissolve Aleuria Aurantia Lectin (AAL) in carbonate buffer solution with pH=9.6 and dilute it to 3 μg / mL, add it into each polystyrene reaction well of a 96-well plate, and make It covered the entire bottom of the well, and 100 μL of the lectin solution was added to each well, and left overnight at 4°C.

[0047] 2) The next day, the lectin solution in the wells was discarded, and 200 μL of blocking solution was added to each well to block the parts not bound to the lectin, and incubated at room temperature for 1 h.

[0048] 3) Wash the 96-well plate 4 times with washing buffer. The washing buffer should fill up the entire reaction well each time, shake gently for 3 minutes, discard the liquid in the well and pat dry on absorbent paper.

[0049] 4) Serum samples were mixed with PBS (pH=7.4) containing 0.01g / mL BSA at 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1: 2560-fold dilutions were added to 96-well plates, 100 μL was added to each well, and incubated at room...

Embodiment 3

[0057] 1) Dissolve and dilute Phytolacca americana (PWM) with pH = 9.6 carbonate buffer to 3 μg / mL, and add it to each polystyrene reaction well of a 96-well plate so that it covers the entire At the bottom of the well, add 100 μL of the lectin solution to each well, and leave overnight at 4°C.

[0058] 2) The next day, the lectin solution in the wells was discarded, and 200 μL of blocking solution was added to each well to block the parts not bound to the lectin, and incubated at room temperature for 1 h.

[0059] 3) Wash the 96-well plate 4 times with washing buffer. The washing buffer should fill up the entire reaction well each time, shake gently for 3 minutes, discard the liquid in the well and pat dry on absorbent paper.

[0060] 4) Serum samples were mixed with PBS (pH=7.4) containing 0.01g / mL BSA at 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1: 2560-fold dilutions were added to 96-well plates, 100 μL was added to each well, and incubated at room temperature for 1 ...

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Abstract

The invention discloses a kit for detecting protein glycosylation subtype and a detection method thereof. The detection kit comprises agglutinin, confining liquid, a biotin labeled antibody, horse radish peroxidase labeled streptavidin, a 3',3',5',5'-tetramethyl benzidine substrate developing solution, a stop solution and a cleaning buffer solution. The detection method comprises the following steps: fixing the agglutinin onto a polystyrene solid phase carrier to be used for recognizing and binding glycoprotein in the sample based on specific recognition of agglutinin and an oligosaccharide chain, recognizing the glycoprotein needing to be detected by virtue of specific binding of the protein and the antibody thereof, and realizing semi-quantitative analysis of the glycoprotein. According to the detection kit and the detection method disclosed by the invention, semi-quantitative detection of single protein glycosylation subtype can be realized, and the kit is easy and convenient to operate and low in cost.

Description

technical field [0001] The invention belongs to the technical field of detection of proteins and their post-translational modifications, and in particular relates to a detection kit for protein glycosylation subtypes and a detection method thereof. Background technique [0002] Glycosylation is one of the most common protein post-translational modifications, and more than half of mammalian proteins are glycosylated. By affecting the solubility, folding, positioning, and maintenance of conformation of proteins, it can regulate the function and degradation of proteins, thereby mediating the interactions between molecules, between molecules and cells, and between cells. In recent years, the research on protein glycosylation in tumors has been increasing. A large number of studies have shown that protein glycosylation will change and have corresponding functional roles in the process of malignant transformation, migration and invasion of tumor cells. In the field of clinical d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 梁一倩陈明伟
Owner THE FIRST AFFILIATED HOSPITAL OF MEDICAL COLLEGE OF XIAN JIAOTONG UNIV
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