Encyclopedia of Experiments: Immunology
Un abonnement à JoVE est nécessaire pour afficher ce contenu. Connectez-vous ou commencez votre essai gratuit.
Transcript
α-galactosidase A and acid α-glucosidase are lysosomal enzymes crucial for cellular metabolism.
α-galactosidase A breaks down substrates such as glycolipids, while acid α-glucosidase hydrolyzes glycogen.
To measure their enzyme activity in vitro, take multi-well plate wells with samples containing known concentrations of α-galactosidase A and acid α-glucosidase, respectively.
Add the required volume of acidic solutions of synthetic 4-Methylumbelliferyl substrates to the wells, specific fluorogenic substrates designed to mimic natural substrates for these enzymes.
Incubate in the dark.
α-galactosidase A and acid α-glucosidase enzymes cleave their respective substrates, releasing the fluorescent product, 4-methylumbelliferone.
Add an alkaline buffer to terminate the enzymatic reaction.
Using a microplate fluorescence reader, measure the fluorescence intensity of the 4-methylumbelliferone in the wells, indicative of its concentration, to determine the α-galactosidase A and acid α-glucosidase enzyme activity, respectively.
