| Assay Method Information | |
| | Biochemical Rat MASP1 Assay |
| Description: | Recombinant rat 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 4 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 40 μM solution of the FRET substrate Dabcyl-MYGGARRL-Glu(Edans)-NH2; (Dabcyl-4-((4-(dimethylamino)phenyl)azo)benzoic acid; Edans-5-[(2-Aminoethyl) amino]naphthalene-1-sulfonyl; 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 340 nm and emission wavelength of 490 nm. IC50 values were calculated from percentage of inhibition of rat MASP2 activity as a function of test compound concentration. |
| Affinity data for this assay | |
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