| Assay Method Information | |
| | Pharmacodynamic Experiment (TPK Enzyme Activity Test) |
| Description: | The purpose of this experimental example was to test the promoting effect of compounds on TPK enzyme activity and evaluate the in vitro activity of the compounds based on EC50 and Emax.1.2.2 Experimental Procedures1.1) The constant-temperature water-bath shaker was pre-heated half an hour in advance at a pre-heating temperature of 37° C.1.2) The stock solution of the compound to be tested was taken out, allowed to thaw at room temperature, and then diluted to the required concentration.1.3) The required reagents were placed on ice to thaw.2.1) For each reaction system, TPK enzyme solution, Tris-HCl buffer solution, and ATP solution were added. The concentration of ATP used was 1-500 mM. The above three solutions were formulated into a homogeneous mixture.2.2) The mixture of the above three solutions was dispensed into centrifuge tubes. Then, the compound at the required concentration was added to each centrifuge tube. Finally, thiamine solution (1-100 M) was added. After the addition of the above system was completed, the centrifuge tubes were tightly capped, inserted into a floating board, and placed in the constant-temperature water-bath shaker that had been pre-heated to 37° C. for incubation for 0.5-2.0 hours.2.3) After the incubation was completed, perchloric acid stop solution was added and mixed well. Samples were taken into 1.5 mL centrifuge tubes and stored at −20° C.3.1) The samples stored at −20° C. were taken out, thawed at room temperature, and then subjected to derivatization treatment. The samples were placed in centrifuge tubes, potassium ferricyanide derivatization reagent was added, and finally phosphoric acid stop solution was added to stop the reaction.3.2) The derivatized samples were placed in sample vials for liquid chromatography analysis, and the contents of TDP/thiamine were detected by high performance liquid chromatography. TPK enzyme activity=TDP (nM)/mg protein/min. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |