Peptoid targeting Abeta42 fibrosome as well as preparation method and application thereof
A type and response technology, applied in the field of biomedicine, can solve the problems of high damage, lack of early detection methods and high cost of cerebrospinal fluid examination, and achieve the effect of strong sensitivity, high affinity and high accuracy
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Embodiment 1
[0052] Example 1 The peptoids of the present invention are synthesized by solid-phase subunit synthesis
[0053] This experimental example provides a peptoid of the present invention and its synthesis method.
[0054] The specific method is as follows:
[0055] 1. Resin swelling: Take 1 g of Rink Amide resin and place it in a synthesis tube, add excess DMF and shake it at room temperature for 1 hour, add 20% hexahydropyridine solution for deprotection, and wash with DCM and DMF alternately for 3 times.
[0056] 2. Coupling of cysteine (cys): Weigh 10 times excess Cys according to the amount of resin, then mix it with HBTU in an equimolar ratio and dissolve it in 0.4mol / L NMM DMF solution. After fully dissolving Put it into a synthesis tube filled with resin, place it on a shaker and shake it for 1 hour, add 20% hexahydropyridine solution for deprotection, and wash with DCM and DMF alternately for 3 times.
[0057] 3. Subsequent subunit connection: According to the amount o...
Embodiment 2A
[0070] Example 2 Preparation of Aβ42 oligomers and fibrils
[0071] In this example, Aβ42 oligomers and fibrils were prepared. Specific steps are as follows:
[0072] (1) Monomerization treatment: In a fume hood, dissolve 2mg of Aβ42 monomer powder (purchased from Jill Biochemical (Shanghai) Co., Ltd.) in 1ml of cooled HFIP (hexafluoroisopropanol), sonicate for 30min, and incubate at room temperature After 2 hours, the Aβ42 was fully dissolved and placed in a fume hood for volatilization. After air-drying, a transparent Aβ peptide film is formed and stored in a -20°C refrigerator until use;
[0073] (2) Preparation of oligomers: the monomerized Aβ42 was added with 1% DMSO (dimethyl sulfoxide) to help dissolve it, and dissolved in PBS solution. Then centrifuge at 12000rpm at 4°C for 30min, and take the supernatant. current use;
[0074] (3) Fibrous body preparation: Add 1% DMSO (dimethyl sulfoxide) to aid dissolution of the monomerized Aβ42, dissolve it in PBS solution, pl...
Embodiment 3
[0077] Example 3 Binding ability test between peptides and β-amyloid polypeptide Aβ42 oligomers and fibrils
[0078] In this example, the peptoids prepared in Example 1 and the binding ability of Aβ42 oligomers and fibrils prepared in Example 2 were tested.
[0079] Specific steps are as follows:
[0080] 1. Preparation of the peptoid chip: pipette the peptoid solution prepared in Example 1 and drop it on the designated area of the chip for SPRi detection. The volume of each spot solution is 0.3ul, and three spots are repeated for each sample.
[0081] 2. Incubation: Place the printed chip in a wet box and incubate overnight in a refrigerator at 4°C;
[0082] 3. Cleaning: Wash the incubated chip with 10×PBS for 10 minutes, 1×PBS for 10 minutes, and deionized water twice for 10 minutes;
[0083] 4. Sealing: The chip is completely submerged in 5% skimmed milk solution (diluted in PBST), and sealed in a refrigerator at 4° C. for more than 6 hours. Then repeat the previous cl...
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