| Assay Method Information | |
| | PDGFRβ HTRF Assay |
| Description: | Assay in white ProxiPlate 384-wellStep 1. Dispensing inhibitors/DMSO and low control: Using the ECHO 555 acoustic dispenser, spot desired compound serial dilutions in DMSO, NEAT DMSO to represent the uninhibited enzyme control, and 10 μM final [imatinib] to the represent the 100% inhibited enzyme controlStep 2. PDGFRβ E+I pre-incubation: Add 2 μL 2× protein solution to columns 1-24 using the Multidrop Combi. Centrifuge at 1000 rpm for 1 min. Incubate 30 min at RTStep 2. Enzymatic reaction: Add 2 μL substrate solution to columns 1-24 to initiate the reaction using the Multidrop Combi; cover/seal the assay plate to reduce evaporation. Centrifuge at 1000 rpm for 1 min. Incubate at room temperature for 3 hours.Final concentrations of components in PDGFRp cascade assay:50 mM Hepes, pH 7.510 mM MgCl20.01% Brij-351 mM EGTA2 mM DTT0.01% Ovalbumin50 μM inactive PDGFRβ0.5 μM TK-substrate biotin peptide62.5 nM SA-XL-665TK antibody-Eu3+-cryptate (diluted by 1/3 final from stock)800 μM ATP≤1% DMSOStep 3. Quench/Detection: Add 2 μl 3× quench/detection solution to columns 1-24 using the Multidrop Combi; cover/seal the plate. Centrifuge 1 min 1000 rpm. Incubate at RT for 60 min. Read the plate in PHERAstar (or similar instrument) on HTRF setting at excitation 337 nmdual emission665/620 nm ratio. |
| Affinity data for this assay | |
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