| Assay Method Information | |
| | Enzyme Activity Assay |
| Description: | Cleavage reactions of the human complement factor B protein: The recombinant human complement factor D protein was diluted 10-fold with PBS (pH 7.4) and stored on ice for later use. The recombinant human complement factor D protein, the recombinant human complement factor B protein, and CVF were added to a reaction buffer (PBS pH 7.4, 10 mM MgCl2, 0.05% CHAPS) to final concentrations of 300 nM, 1 μM, and 1 μM, respectively. After they were well mixed, the mixture was reacted in a constant-temperature incubator at 37° C. for 3 h to give a complex of CVF and the post-cleavage fragment Bb of the recombinant human complement factor B protein (hereinafter referred to as CVF:Bb). A 100 mM Na2CO3 solution and a 100 mM NaHCO3 solution were prepared and mixed in a ratio of Na2CO3 to NaHCO3 of 3:7 (v/v) to adjust the pH to 9.5. The mixture was stored at room temperature for later use. 20 mM test compounds dissolved in 100% DMSO were serially diluted with 100% DMSO to 2000, 500, 125, 31.25, 7.8125, 0.488281, 0.12207, 0.030518, and 0.007629 M, with the blank well containing 100% DMSO. The compounds and 100% DMSO were then 20-fold diluted in C3 reaction buffer (PBS pH 7.4, 1 mM MgCl2, 0.05% CHAPS). |
| Affinity data for this assay | |
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