| Assay Method Information | |
| | Bioactivity Assay |
| Description: | Preparation and Treatment. Stock Solution Preparation. A 10 mM DMSO stock solution was formulated for each compound. A 30 mM DMSO stock solution was formulated for positive control HA130. Preservation: The DMSO stock solutions were stored at room temperature in a desiccator for a short period of time or at −20° C. for a long period of time. Preparation All compounds were serially 3-fold diluted in DMSO from 10 μM to the 10th concentration.A 1000× positive control (10 mM, HA130) and a 1000× negative control (100% DMSO) were prepared. The plates were shaken for 5 min. Preparation of 1× Reaction Buffer:>5× reaction buffer and cold water were mixed well in a volume ratio of 1:4. Preparation of 20 U/mL Choline Oxidase Stock Solution:1 tube of choline oxide in a kit was dissolved in 1× reaction buffer. The aliquots were frozen at −20° C. Preparation of 200 U/mL Horseradish Peroxidase (HRP) Stock Solution:1 tube of HRP in a kit was dissolved in 1 mL of 1× reaction buffer. The aliquots were frozen at −20° C. Preparation of 20 mM Amplex Red Reagent (for Detection) Stock Solution Amplex Red reagent and DMSO were equilibrated at room temperature. 1 tube of Amplex Red reagent in a kit was dissolved in 200 μL of DMSO.Detection: a) 2×ATX (2 ng/L), 2×HRP (2 U/L), 2× choline oxidase (0.2 U/mL) were added to 1× reaction buffer. b) 20 μL of the reaction mixture was added to the plate (PE, 6007270) (refer to step a). c) 40 nL of diluent was added to the plate using Echo (refer to Preparation ). d) 2×LPC (16:0) (60 mM) and 2× Amplex Red (400 M) were prepared using 1× reaction buffer. e) 20 μL of the mixture was added to the plate (refer to step d). f) The plate was shaken for 30 s and allowed to stand at room temperature for 30 min. g) The plate was examined using Envision, and the procedure was as follows: Excitation at 530 nm; and Emission at 590 nm. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |