In Vitro Measurement of α-Galactosidase A and Acid α-Glucosidase Enzyme Activity

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…

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