To verify the affinity and kinetics between sitagliptin and the novel target, we performed this experiment using Biacore T200 (GE Healthcare) instrument based on SPR, and used GraphPad Prism 8.0 software to visualize the data results (18 (link), 19 (link)).
Firstly, the amino acid sequence of the target protein was searched by Uniprot database, and the sequence was imported into Expasy (https://web.expasy.org/ ) website to calculate the isoelectric point of the protein (20 (link)). Then, the preconcentration experiment was carried out to determine the optimal coupling conditions. The target protein was coupled to CM5 chip (GE Healthcare) using the Immobilization module in Biacore T200 Control Software, and the reference channel was set to deduct the background. The chip was activated by EDC/NHS (GE Healthcare), and the uncoupling site was blocked by ethanolamine (GE Healthcare).
sitagliptin (HPLC ≥ 98%, Shanghai yuanye Bio-Technology Co., Ltd) was dissolved in DMSO (VWR) solution and diluted with PBS-P+ (GE Healthcare) solution to the desired compound concentration (1×PBS-P+, 5% DMSO). The affinity and kinetics of sitagliptin with the novel target protein were tested by LMW kinetics module in Biacore T200 Control Software, and extra wash after injection with 50% DMSO was added.
Firstly, the amino acid sequence of the target protein was searched by Uniprot database, and the sequence was imported into Expasy (
sitagliptin (HPLC ≥ 98%, Shanghai yuanye Bio-Technology Co., Ltd) was dissolved in DMSO (VWR) solution and diluted with PBS-P+ (GE Healthcare) solution to the desired compound concentration (1×PBS-P+, 5% DMSO). The affinity and kinetics of sitagliptin with the novel target protein were tested by LMW kinetics module in Biacore T200 Control Software, and extra wash after injection with 50% DMSO was added.
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