| Assay Method Information | |
| | MIF Enzymatic Assay |
| Description: | Assays were performed in small-volume clear-bottom black 96 or 384-well polystyrene plates (Greiner Bio-One). First, 2 μL of the enzyme solution containing 6 nM of MIF in DPBS, 0.025% w/v BSA, and 300 μM CHAPS was dispensed onto sample and negative control wells using Multidrop Combi with metallic tip cassettes (Thermo Fisher Scientific) previously treated with Sigmacote. Then, 2 μL of the same buffer without MIF was dispensed onto positive control wells. The reaction was started by addition of the following to all wells: 2 μL of substrate solution containing 3 mM ketonic pHPP in 200 mM boric acid, 25 mM sodium phosphate, 0.025% w/v BSA, and 300 μM CHAPS at pH 6.0. In order to remove bubbles, the plate was centrifuged in an Allegra 25R centrifuge (Beckman Coulter, Inc., Brea, CA) at 1000 rpm for 2 min at room temperature. Then, the plate was read in an EnVision. Final concentrations of enzyme and substrate were 3 nM and 1.5 mM, respectively. Initial rates were calculated for each well as the slope of the absorbance progress curve. |
| Affinity data for this assay | |
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