| Assay Method Information | |
| | In Vitro Screening Experiment |
| Description: | 1. Reagent Preparation:1) Preparation of 1× AlphaLISA test buffer solution:2.5 ml of 10× AlphaLISA test buffer solution was added to 22.5 ml of Milli-Q water to produce the 1× AlphaLISA test buffer solution.2) Preparation of mixture (25 μg/mL/2.5 nM) of 2.5× AlphaLISA Acceptor beads and Biotinylated Antibody Anti-PCSK9:50 μL of 5 mg/mL Alpha LISA Anti-PCSK9 Acceptor beads and 50 μL of 500 nM Biotinylated Antibody Anti-PCSK9 were added to 9,900 μL of 1× AlphaLISA test buffer solution.3) Preparation of 2× Streptavidin (SA) Donor beads (80 μg/mL):200 μL of 5 mg/mL SA-Donor beads was added to 12,300 μL of 1× AlphaLISA test buffer solution.2. Operation Steps:1) DMSO was added to the test compound to produce the 50 μM stock solution, which was then serially diluted into 10 concentrations by 3-fold, and placed in the 384-pore plate;2) PCSK9-Huh7 cells were added in the 384-pore plate, and the cell plate was cultured in the incubator for 20 h;3) 20 μL/pore mixture of 2.5× AlphaLISA Acceptor beads and Biotinylated Antibody Anti-PCSK9 was added, and incubated at 23° C. for 60 min.4) 25 μL/pore 2× Streptavidin (SA) Donor beads were added; and incubated for 30 min at 23° C. (kept in dark place).5) EnVision-Alpha Reader reading:Test condition: Total Measurement Time: 550 ms, Laser 680 nm Excitation Time: 180 ms, Mirror: D640as, Emission Filtered: M570w, Center Wavelength 570 nm, Bandwidth 100 nm, Transmittance 75%.3. Data Analysis:Date was analyzed with Prismsoftware (nonlinear regression (Sigmoidal dose-response-variable slope)Inhibition ratio %=100*(Sample reading−LC reading)/(HC reading−LC reading). |
| Affinity data for this assay | |
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