| Assay Method Information | |
| | LOXL2 Enzyme Activity Assay (20 minutes) |
| Description: | The following reagents were prepared:Reagents500 mM CHES, pH 9 (with NaOH), filtered (MW=207.29)20.7 g in 200 ml in dH2O10% Pluronic F-127 (store at 4° C.)1 g in 10 ml in dH2O10% BSA (store at 4° C.)−2.5 g in 25 ml dH2O1 M MgCl2.6H2O (MW=203)5.1 g in 25 ml dH2O5 M Urea (MW=60.06)60 g in 200 ml dH2O5 M NaCl (MW=58.44)14.6 g in 50 ml dH2O8.5 M Cadaverine Dihydrochloride (MW=175.10): Sigma, Cat No: C85611.49 g in 1 ml dH2OPromega ROS-Glo H202 Assay Kit, 50 mlCat No. G8821DMSOLOXL2 Assay Buffer: Volume (μl) Reagent 1 × plate Final Concn 500 mM CHES, pH 9 5000 100 mM 10% Pluronic F-127 125 0.05% 10% BSA 1250 0.5% 1M MgCl2 25 1 mM 5M Urea 5000 1M 5M NaCl 500 100 mM dH2O 13100 — Total Volume 25 000 —Inhibitors (Test Compounds):10 mM stock diluted to 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, and 0.0003 mM in drug plate, resulting in a concentration of 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003 uM in the assay.LOXL2:LOXL2 batch is obtained from R&D systems/Bio-techneAssay Plates: 96 well white polystyrene, flat bottom plates, no lid: Fisher, Cat No. DPS-134-050AProcedure:1. Dilute LOXL2 enzyme in Assay Buffer (˜4 ml for one plate, 8 ml for two plates) (dilution dependent on batch activity)1. Add 0.5 μl test compound serial dilutions, in duplicate2. Add 0.5 μl serial dilutions of positive control, BAPN, down column 11 (no duplication)3. Add following controls: 0.5 μl DMSO (100% activity control)4. Cover plate and incubate for 20 minutes (Method (a) in Table 1), 60 minutes (Method (b) in Table 1) or 20 hours (Method (c) in Table 1), at room temp on a plate shaker5. Prepare Start Mix: Volume Reagent 1 × plate 2 × plate Final ConcnAssay Buffer 2 ml 3 ml —8.5M Cadaverine 23 μl 34.5 μl 97.8 mMH2O2 Substrate (1:80) 25 μl 37.5 μl 125 μM6. Add 10 μl Start Mix to all wells7. Incubate for 60 min at room temp on a plate shaker8. Prepare Detection Reagent: Volume Reagent 1 × plate 2 × plate Luciferin 5 ml 10 ml D-cysteine (1:100) 50 μl 100 μl Signal Enhancer (1:100) 50 μl 100 μl9. Add 50 μl Detection Regent to each well EXCEPT No Luciferin controls (add 50 82 l assay buffer)10. Incubate for 20 min at room temp on a plate shaker, protected from light11. Measure luminescence using a plate reader (integration 500 ms)Values for the blank (no LOXL2 controls) are subtracted from all samples. The DMSO controls are set as 100% activity and samples are calculated as a % of the DMSO control. Data is plotted using Graph pad Prism software, and a non-linear regression line is calculated using a variable slope sigmoidal dose response equation.:Y=Bottom+(Top-Bottom)/(1+10{circumflex over ( )}((LogIC50−X)*HillSlope)).Where X is the logarithm of concentration and Y is the response. The IC50 is the concentration of the drug that produces a percentage control fluorescence value midway between the saturation and zero effect plateaus. Two independent assays are usually performed, and the mean IC50 is reported. |
| Affinity data for this assay | |
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