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A kind of acidic polysaccharide electrophoresis standard product and its preparation method and application

A standard product, polysaccharide technology, applied in the field of acid polysaccharide electrophoresis standard product and its preparation, can solve the problem of low sensitivity, achieve the effects of improving sensitivity and accuracy, improving experimental efficiency and saving experimental time

Active Publication Date: 2020-06-30
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Agarose electrophoresis is a simple method for determining the purity and molecular weight of polysaccharides, but it requires steps such as precipitation, staining, and rinsing, and the sensitivity is low

Method used

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  • A kind of acidic polysaccharide electrophoresis standard product and its preparation method and application
  • A kind of acidic polysaccharide electrophoresis standard product and its preparation method and application
  • A kind of acidic polysaccharide electrophoresis standard product and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] A kind of preparation method of acidic polysaccharide electrophoresis standard substance, comprises the steps:

[0057] (1) Synthesis of hyaluronic acid-tyramine: Take a beaker as a container, take 0.5g of hyaluronic acid with three molecular weights of 8kDa, 200-400kDa, and 700-900kDa, and dissolve them in 100mL of phosphate buffer with a pH of 7.4 In the solution, fully dissolve at 4°C for 12 hours, add 2g of Tyramine Tyr, mix and dissolve thoroughly, then seal the beaker with plastic wrap and place it in a shaker at 150r / min, react at 37°C for 24h; then add 1g of cyanide Based on sodium borohydride, seal the beaker with plastic wrap and place it in a shaker at 150r / min, react at 37°C for 96h, centrifuge the reaction solution at 4000rpm, take the respective supernatants, and obtain three transparent Hyaluronic acid-tyramine solution;

[0058] (2) Synthesis of hyaluronic acid-tyramide-FITC: add 250mg fluorescein isothiocyanate (FITC) respectively to the three kinds of...

Embodiment 2

[0061] Aminonaphthalenetrisulfonic acid (ANTS) labeled hyaluronic acid (HA), comprising the following steps:

[0062] (1) Weigh 100 mg of hyaluronic acid with a molecular weight of 200-400 kDa and dissolve it in 100 mL of water;

[0063] (2) Prepare 0.15M ANTS-acetic acid solution and 1M NaBH 3 CN-DMSO solution, mix 1mL hyaluronic acid solution and 1mL ANTS-acetic acid solution, react at room temperature for 1h in the dark, then add 1mL NaBHCN-DMSO solution, place the mixed solution in a 37°C water bath for 18h, centrifuge, take The supernatant was obtained as a hyaluronic acid-ANTS (HA-ANTS) solution, which was stored in the dark at 4°C until electrophoresis detection;

[0064] (3) Fluorescence-assisted polysaccharide electrophoresis (FACE)

[0065] 1) Reagent preparation

[0066] ①Separating gel stock solution 40% acrylamide mixture: 38% (w / v) acrylamide + 2% (w / v) bisacrylamide, pH<7.

[0067] Acrylamide (MW121.14) 38g;

[0068] Methylenebisacrylamide (MW75.07) 2g;

...

Embodiment 3

[0114]The comparison of hyaluronic acid without tyramine and tyramine-labeled hyaluronic acid includes the following steps:

[0115] Option 1: The preparation of hyaluronic acid + FITC includes the following steps:

[0116] (1) Prepare phosphate buffer (PH=7.4);

[0117] (2) Take hyaluronic acid with a molecular weight of 1400kDa, dissolve it in 1% acetic acid to prepare a 0.01g / mL hyaluronic acid solution, adjust the pH to 9.0±0.1 with 0.1g / mL NaOH, and then place it at 37°C and 150r / min Constant temperature shaking incubator, constant temperature shaking for 30min;

[0118] (3) Prepare 10mg / mL FITC-DMSO solution, mix 10mL hyaluronic acid-acetic acid flocculent solution with 1mL FITC-DMSO solution, shake the mixed solution overnight in the dark, and then centrifuge the mixed solution at 4000rpm for 15min , take the precipitate;

[0119] (4) Dissolve the precipitate in 10 mL of pure water, add 3 times the volume of the solution in absolute ethanol, react overnight, centrifu...

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Abstract

The invention belongs to the technical field of fluorescently labeled polysaccharides, and in particular relates to an acidic polysaccharide electrophoresis standard product and its preparation method and application, including: 1) Synthesis of hyaluronic acid-tyramine: take 8000, 20-400,000, 70-900,000 Hyaluronic acid with three molecular weights was dissolved in alkaline phosphate buffer solution respectively, after dissolution, tyramide Tyr was added for reaction, and then sodium cyanoborohydride was added for reaction. After completion, the reaction solution was centrifuged, and the respective upper Clear liquid, obtain three kinds of hyaluronic acid-tyramide solutions; 2) Synthesis of hyaluronic acid-tyramide-FITC: add isothiocyanate fluorescence respectively in three kinds of hyaluronic acid-tyramide solutions prepared in step 1) After the reaction is completed, three kinds of hyaluronic acid-tyramide-FITC solutions are obtained. The hyaluronic acid with different molecular weights marked by FITC in the present invention can completely become a marker in polysaccharide labeling, and can quickly detect whether the polysaccharide labeling is successful or not.

Description

technical field [0001] The invention belongs to the technical field of fluorescently labeled polysaccharides, and in particular relates to an acidic polysaccharide electrophoresis standard product and its preparation method and application. Background technique [0002] The information disclosed in the Background of the Invention is only intended to increase the understanding of the general background of the invention, and is not necessarily to be taken as an acknowledgment or any form of suggestion that the information constitutes the prior art that is already known to those skilled in the art. [0003] The 21st century is an important period for the development of glycochemistry and glycobiology. With the continuous development of science, polysaccharides play an increasingly important role in food and medicine. Polysaccharides are a complex class of compounds. Polysaccharides with different molecular weights have different biological activities, and even some polysacchari...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/28G01N27/447G01N27/416
CPCG01N1/28G01N27/4163G01N27/447G01N2001/2893
Inventor 宋淑亮张娥王宇吉爱国
Owner SHANDONG UNIV