In Vitro Measurement of α-Galactosidase A and Acid α-Glucosidase Enzyme Activity
Published: February 29, 2024
Abstract
Source: Lukas, J., et al. In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease. J. Vis. Exp. (2017)
This video demonstrates the in vitro measurement of enzyme activity for α-galactosidase A and acid α-glucosidase in samples using synthetic 4-Methylumbelliferyl substrates.
Protocol
1. Cell Harvest and α-galactosidase A or Acid α-glucosidase Activity Measurement Cell harvest On the day of the harvest, remove the cells from the incubator and aspirate the medium. Carefully wash the cells 2 times with PBS with Ca2+ and Mg2+. NOTE: This step is critical because DGJ and DNJ are potent inhibitors of α-galactosidase and α-glucosidase, respectively, and any leftover would inval…
Declarações
The authors have nothing to disclose.
Materials
| Sodium chloride | Merck, Darmstadt, Germany | 1,06,40,41,000 | |
| Potasium chloride | Merck, Darmstadt, Germany | 1,04,93,60,500 | |
| MgCl2 x 6H2O | Merck, Darmstadt, Germany | 1,05,83,30,250 | |
| D (+)-Glucose monohydrate | Merck, Darmstadt, Germany | 1,08,34,22,500 | |
| Sodium Hydroxide | Merck, Darmstadt, Germany | 1,06,49,80,500 | |
| Sodium acetate | Merck, Darmstadt, Germany | 1,06,26,40,050 | |
| HEK293H cell line | Invitrogen, Karlsruhe, Germany | 11631-017 | |
| SafeSeal reaction tubes, 1.5mL | Sarstedt, Nümbrecht, Germany | 72,706 | |
| Tecan GENios Reader | Tecan, Männedorf, Switzerland | ||
| Magellan data analysis software v6.6 | Tecan, Männedorf, Switzerland | N.A |
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In Vitro Measurement of α-Galactosidase A and Acid α-Glucosidase Enzyme Activity. J. Vis. Exp. (Pending Publication), e21997, doi: (2024).