Method for quantitatively detecting sugar chain structures in glycosphingolipid
A quantitative detection and glycosphingolipid technology, which is applied in the preparation of test samples, measurement devices, color/spectral characteristics measurement, etc., can solve the problems of cumbersome operation, long experiment cycle and high cost
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Embodiment 1
[0045] In order to better analyze the spectrum of glycolipid sugar chains, 37 kinds of lectins that recognize different sugar chain segment structures are dotted into a 4×10 matrix along with the negative control BSA and the position-labeled Cy3 labeled BSA.
[0046] GM1 lipo chain digestion and purification
[0047] First, dissolve 400μMGM1 in the digestion reaction buffer. The digestion buffer is 0.8% TDC, 50mM NaAc, pH5.5. Add 40mUSCDase enzyme. After mixing, add 200μL of decane to remove the fatty acid chains produced by the digestion reaction. The reaction was shaken at low speed for 24h on a shaker at ℃, and decane was replaced every 4-6h during the period. After 24h, the upper decane was aspirated and discarded, and the SCDase enzyme was denatured by heating in a water bath at 100℃ for 5min, then centrifuged at 12000rpm for 15min, and the supernatant was taken. After evaporating to dryness, the digested GM1 can be obtained.
[0048] Purification and quantification of GM1 afte...
Embodiment 2
[0067] This method was used to verify the glycolipid and sugar chains of the liver cancer cell line HePG2.
[0068] 1) Preparation of lectin chip:
[0069] 1.1) Take the epoxidized film base for use;
[0070] 1.2) Prepare the lectin into a spotting solution with a concentration of 1 mg / ml;
[0071] 1.3) Add the prepared spotting solution to the 384-well plate; then use the Beijing Boao Crystal Core 48 spotting system to spot the epoxidized substrate;
[0072] 1.4) Incubate the prepared chips for 12 hours in an environment with a humidity of 60%;
[0073] 1.5) Vacuum the incubated chip at 37°C for 3 hours to dry the chip and fix the lectin on the chip;
[0074] 1.6) Place the fixed lectin chip in a 4°C desiccator for use.
[0075] 2) Preparation of glycolipid sample target for lectin chip:
[0076] 2.1) Separation and purification of glycosphingolipids Collect 3×106 human liver cancer cell line HepG2 cells, 3mL chloroform / methanol (2:1v:v), 3mL chloroform / methanol (1:1v:v) to crudely extract...
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