| Assay Method Information | |
| | Inhibition of rhGAA Determined by Fluopol-ABPP |
| Description: | The inhibiting activity on recombinant human acid α-glucosidase (rhGAA) of the compounds is implemented using the Fluopol-ABPP method (Fluorescence Polarization Activity Based Protein Profiling) described in the publication of Lahav et al., 2017, J. Am. Chem. Soc. 139: 14192-14197. This technique, based on the competition between an inhibitor and a fluorescent probe capable of binding covalently to the active site of an enzyme, makes it possible to measure the affinity of this inhibitor for the active site of the rhGAA enzyme used for these experiments by the laboratory of Professors Herman S. Overkleeft and Johanes M. F. G. Aerts, Leiden Institute of Chemistry, Leiden University (NL), is the enzyme marketed under the name Myozyme . The median inhibiting concentrations (IC50) are determined in the Mcllvaine buffer (citrate-phosphate) 150 mM at pH 5.0, in the presence of 0.1% bovine gamma-globulin (p/v) and 0.5 mg/mL of Chaps detergent (Sigma) in 96-well plates (Griener). The rhGAA enzyme (10 μg/mL) is pre-incubated with solutions of inhibitor (containing 2.5% DMSO that was used to prepare the mother solutions of the compounds) at various concentrations [I] in the buffer, for 45 minutes at 37 C. The tetraaminomethylrhodamine (TAMRA) fluorescent probe in solution (25 nM) in the buffer is next added to the mixture. After 4 hours, the samples are irradiated with a polarized light (λ=530 nm) and the fluorescence emitted (λ=580 nm) is measured by means of an Infinite M1000Pro (Tecan) spectrofluorimeter. |
| Affinity data for this assay | |
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