Isoelectric focusing electrophoretic method of hemopoietin

A technology of isoelectric focusing electrophoresis and erythropoietin, which is applied in the field of protein electrophoresis, can solve the problems such as the separation of various bands that cannot be EPO, and the high upper limit voltage requirement.

Active Publication Date: 2018-05-04
INST OF ZOOLOGY GUANGDONG ACAD OF SCI +1
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0040] The purpose of the present invention is to: overcome the defects in the prior art that the upper limit voltage is very high ...

Method used

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  • Isoelectric focusing electrophoretic method of hemopoietin
  • Isoelectric focusing electrophoretic method of hemopoietin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0056] 1. Instrument device

[0057] Current meter power supply DYY-8C type, manufacturer: Beijing Liuyi Instrument Factory.

[0058] Electrophoresis instrument Model 111Mini IEF Cell, manufacturer: BIO-RAD.

[0059] Horizontal shaker WD-9405B, manufacturer: Beijing Liuyi Instrument Factory.

[0060] 2. Reagent and test solution preparation

[0061] (1) Water (resistivity not less than 18.2MΩ.cm).

[0062] (2) 25wt% acrylamide: Weigh 24.25g of acrylamide and 0.75g of N,N'-methylenebisacrylamide, add an appropriate amount of water to dissolve, and dilute to 100mL, filter out impurities with a 0.45μm filter, and put the brown reagent Store in the bottle at 2-8°C, valid for 6 months.

[0063] (3) 10.5M urea: Weigh 31.53g urea and dissolve it in an appropriate amount of water, and finally add water to 50mL.

[0064] (4) 60wt% sucrose: Weigh 6g of sucrose and dissolve it in 5mL of water, and finally add water to 10mL.

[0065] (5) 0.1wt% FMN-Na solution: 100mg FMN-Na was diss...

experiment example

[0088] sample for Erythropoietin, the product name is Darbepoetin Afla, the manufacturer is KyowaHakko Kirin Co., Ltd, the batch number is 16802E BPR1610005, the production date is 2016-08-01, and the validity period is 2018-07-31. The isoelectric point standard product (Marker) has a PI range of 3.5-10.7, the manufacturer is SERVA, and the product number is 39212.01. Electrophoresis results such as figure 1 and figure 2 shown.

[0089] figure 1 EPO was separated by isoelectric focusing electrophoresis according to the isoelectric focusing method provided in the instruction manual of BIO-RAD's 111-type current meter. Its gel-making formula is: 2.75mL of water, 1.0mL of 25% acrylamide solution, 1.0mL of 25% glycerin, 0.183mL of 40% pH2-4 ampholyte solution, 0.075mL of 40% pH3-10 ampholyte solution, and the catalyst solution is 0.1% FMN-Na 25 μl, 10% AP 7.5 μl, TEMED 1.5 μl.

[0090] figure 2 EPO was separated by isoelectric focusing electrophoresis according to the me...

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Abstract

The invention discloses an isoelectric focusing electrophoretic method of hemopoietin. A BIO-RAD 111 type electrophoresis apparatus is used to carry out isoelectric focusing electrophoresis. Each 5 mLof glue comprises 1 mL of acrylamide monomer solution (25 wt%), 3.33 mL of urea (10.5 M), 0.412 mL of cane sugar (60wt%), 0.183 mL of amphoteric electrolyte solution (40 wt%, pH 2-4), 0.075 mL of amphoteric electrolyte solution (40 wt%, pH 3-10), and a catalyst solution, wherein the catalyst solution is mixed in advance and comprises 25 [mu]L of FMN-Na (0.1 wt%), 7.5 [mu]L of AP (10 wt%), and 1.5[mL] of TEMED. Before sample injection, pre-electrophoresis is performed for 10 minutes under a voltage of 200 V. Electrophoresis is performed through following phases: first phase, 50 V, 30 minutes;second phase, 100 V, 60 minutes; third phase, 200 V, 30 minutes; fourth phase, 450 V, 60 minutes; and fifth phase, 590 V, until the current is zero. The method can separate the hemopoietin charge isomers with different isoelectric points, and has the advantages that the requirement on the upper limit of voltage is not high and no cooling apparatus is needed.

Description

technical field [0001] The invention belongs to the technical field of protein electrophoresis, and more specifically, the invention relates to an isoelectric focusing electrophoresis method capable of separating charge variants of erythropoietin. Background technique [0002] Erythropoietin (EPO for short) in the human body is a hormone-like substance secreted by the kidney and liver, which can promote red blood cell production. It is an acidic glycoprotein containing sialic acid, one of the members of the hematopoietic cytokine superfamily. Undifferentiated stem cells differentiate into erythroid stem cells, where erythropoietin acts to make them preerythroblasts. It can promote further maturation into erythroblasts, reticulocytes, synthesis of hemoglobin and flow into peripheral blood vessels. Generally, in the state of anemia and hypoxia, the supply of erythropoietin will increase according to the oxygen demand of body tissues, but its content is very low in renal anem...

Claims

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Application Information

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IPC IPC(8): G01N27/447
CPCG01N27/44795
Inventor 张钦邓新宇王笑非
Owner INST OF ZOOLOGY GUANGDONG ACAD OF SCI
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