| Assay Method Information | |
| | Enzyme Kinetics - Measuring Inactivation Rates of Inhibitors |
| Description: | Inactivation solutions contained a final concentration of 16.55 nM of Cerezyme (Recombinant GCase) in 200 μL of Reaction Buffer (50 mM acetate, 0.2% v/v Triton X-100, 0.3% w/v sodium taurocholate, pH 5.5). The Cerezyme was collected from leftover patient vials and had approximately 8275 nM in the vial. These enzyme Reaction Buffers were brought to 37 C. and spiked with a corresponding inhibitor to make the final inhibitor concentration either 20 nM, 40 nM, 60 nM, 80 nM, 120 nM, 160 nM and 200 nM. Once the inhibitor was added this was considered time=0 minutes. Meanwhile a 96 well plate inside a Biotek Synergy 4 hybrid multi-mode reader at 37 C. was being incubated. In the wells of the plate were waiting a high concentration (3.2 mM) of 2,4-DNP-β-Glc substrate in 180 μL of Reaction Buffer. As the inhibitor inactivation was occurring, 20 μL aliquots were taken from the inactivation solutions and diluted on the plate containing the substrate bringing the final concentration of the substrate to 4 mM. Once all the aliquots were added the solution in the wells was measured for absorbance at 400 nm for 8 minutes. At this time the measurements were paused and the next set of aliquots were taken to see how residual enzyme activity changes over time. |
| Affinity data for this assay | |
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