Application of Vandetanib to preparation of targeted EphB4 anti-tumor drug
An anti-tumor drug, the technology of vandetanib, applied in the field of medicine, can solve the problem that there is no report on the application of vandetanib in anti-leukemic tumors, and achieve the effect of inhibiting the activity of EphB4 kinase
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Embodiment 1
[0028] Determination of EphB4 expression by Western blotting: Western blotting was used to investigate the expression levels of EphB4 in three different types of leukemia cells. When the cells were in the exponential growth phase, the cells were lysed with RIPA cell lysate for 30 min, and the cell lysate was centrifuged at 12,000 g, 10 min, and 4° C. to precipitate the cells. Aspirate the supernatant and add a certain amount of 5×loading buffer, heat and boil for 5 minutes to denature the protein, mark it, and store it at -20°C for later use. Load the processed protein sample on the prepared 10% SDS-PAGE, add 1× electrophoresis buffer, turn on the power, and maintain a steady voltage of 120mV for sample separation. The separated samples were purposely cut and transferred to the membrane using a steady flow of 120mA. After the membrane transfer, the PVDF membrane was placed in skimmed milk powder blocking solution for blocking for 2 h. The EphB4 primary antibody was then incu...
Embodiment 2
[0031] Kinase assay to investigate the effect of vandetanib on EphB4 kinase activity: the homogeneous time-resolved fluorescence (HTRF) method was used to detect the effect of the drug on inhibiting the phosphorylation of EphB4 kinase. Add 2 μL of EphB4 kinase and 2 μL of substrate to the 384-well plate, then add 4 μL of vandetanib at different concentrations, and finally add 2 μL of ATP to the 384-well plate, and use a shaker to mix the reaction system evenly. Place the 384-well plate in a 37°C oven for incubation. After reacting for 30 minutes, add Eu 3+ -TK-antibody working solution and streptavidin-XL665 each 5μL into a 384-well plate to terminate the reaction. After reacting at room temperature for 1 h, the 384-well plate was put into a multi-functional microplate reader for detection. The detection conditions were as follows: 615nm was selected as the excitation wavelength, and 665nm was selected as the emission wavelength. see image 3 , the results showed that vande...
Embodiment 3
[0033] SPR experiment confirms the target: install an NTA chip in the OpenSPRTM instrument, and then pump the PBS buffer at the maximum flow rate. After reaching the signal baseline, inject 80% IPA (isopropanol) from the sample port, turn the control valve to inject, run for 10s and return to load to remove air bubbles, after reaching the baseline, rinse the sample loop with buffer, and use air emptying. After the signal reaches baseline, adjust the buffer flow rate to 20 μL / min. Introduce 250 μL of imidazole solution into the injection port and inject to initialize the sensor surface, initialize 2-3 times until the curve after injection is consistent. Introduce 250 μL of 40 mM NiCl 2 to the injection port and inject, through the Ni 2+The ions charge the NTA, and the surface is now ready to be functionalized with his-tag ligands. Prepare the His-tag ligand EphB4-1 (50 μg / mL) dissolved in 250 μL running buffer, inject it into the sample well, and interact for at least 5 min...
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