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).

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