| Assay Method Information | |
| | Biochemical Human MASP1 Assay |
| Description: | Recombinant human MASP1 enzyme produced in the HEK 293 cells was diluted in the reaction buffer (50 mM HEPES pH 8,0; 100 mM NaCl; 0,01% CHAPS; 0.5 mM Gluthathione) to the concentration of 20 nM and 25 μl was transferred into each single well of 384-well white microtiter plate (Greiner Bio One 781075). 1 μl of the inhibitor compound solution (dissolved in DMSO, at the corresponding concentration) or pure DMSO as a control was added to the same wells. The enzymatic reaction was initiated by addition of 25 μl of 20 μM solution of the FRET substrate ABZ-MYGGARRL-Lys (Dnp)-NH2; (ABZ-2-aminobenzoyl; DNP 2,4-dinitrophenyl; custom synthesis by Jerini Peptide Technologies, Berlin) in the reaction buffer. The microtiter plate was incubated for 60-120 min at the temperature of 32 C. The increase of fluorescence intensity was measured in appropriate fluorescence plate reader (e.g. TECAN Ultra) using excitation wavelength of 320 nm and emission wavelength of 420 nm. IC50 values were calculated from percentage of inhibition of human MASP1 activity as a function of test compound concentration. |
| Affinity data for this assay | |
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