Acidic polysaccharide electrophoresis standard substance as well as preparation method and application thereof
An acid polysaccharide, standard product technology, applied in the preparation of test samples, material analysis and sampling by electromagnetic means, etc., can solve the problem of low sensitivity, improve sensitivity and accuracy, save experimental time, and improve experimental efficiency. Effect
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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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