| Assay Method Information | |
| | LMPTP Primary Screening Protocol |
| Description: | A listing of materials is provided: Item, source, catalog no. LMPTP-A Enzyme Stock Solution (4.22 mg/ml or 206.8 μM), SBMRI Protein Facility, N/A OMFP, Sigma, M2629-100 MG Bis-Tris pH 6.0, Fisher Sci, BP301-100 Triton-X 100, Sigma, T9284 DTT, Sigma, D9779 Mol. Grade Water, Mediatech, Inc., 46-000-CM 1536-well black High base opaque bottom plate, Nexus Biosystems, 00019120Final assay conditions are: 0.625 nM LMPTP-A Enzyme 40 μM OMFP 50 mM Bis-Tris pH 6.0 1 mM DTT 0.01% Triton-X 100 20 μM test compound 3% DMSO (2% from substrate and 1% from compounds) 6 μL reaction volume 50 minutes incubation at room tempAssay Procedure 1. Prepare Reagents as described in section F. Recipe. 2. Using LabCyte Echo, transfer 60 nL from 2 mM test compound source plate into assay plate Col. 5-48 (final concentration of test compounds is 20 μM). 60 nL of DMSO should be transferred to col. 1-4 for positive and negative control wells. 3. Spin plates at 1000 rpm for 1 minute in centrifuge. 4. Using the Beckman Coulter Bioraptr, add 3 μL/well of control buffer to columns 1 and 2. 5. Using the Bioraptr, add 3 μL/well of enzyme solution to col. 3-48. 6. Using the Bioraptr, add 3 μL/well of substrate solution to col. 1-48. 7. Spin plates at 1000 rpm for 1 minute in centrifuge. 8. Incubate plates in the dark at room temperature for 50 minutes. 9. Read plates on PerkinElmer Viewlux using a FI protocol. |
| Affinity data for this assay | |
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