Encyclopedia of Experiments: Immunology
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Dilute the calculated amount of each sample and pipette it into fresh 1.5-milliliter reaction tubes to obtain 0.05 micrograms of alpha-galactosidase A or 0.5 micrograms of acid alpha-glucosidase per microliter of solution. Vortex the samples for 5 seconds again, and pipette 10 microliters of this dilution and duplicate into a 96-well plate.
Start the reactions by adding 20 microliters of the respective substrate solution. For alpha-galactosidase A, add 2 millimolar 4-methylumbelliferyl-alpha-D-galactopyranoside or 4-MU-Gal in 0.06 molar phosphate citrate buffer, pH 4.7. For acid alpha-glucosidase, add 2 millimolar 4-methylumbelliferyl-alpha-D-glucopyranoside or 4-MU-Glu in 0.025 molar sodium acetate, pH 4.0.
Incubate the enzyme reactions for 1 hour in the dark at 37 degrees Celsius and 300 RPM on an orbital shaker. Then, terminate the reaction by adding 200 microliters of 1.0 molar pH 10.5 adjusted glycine sodium hydroxide buffer. Prepare a standard curve of 4-MU from a 0.01 milligram per milliliter stock according to the text protocol, and pipette 10 microliters of the solutions in duplicate into a 96-well plate.
Add 200 microliters of the 1.0 molar glycine sodium hydroxide buffer to each well in order to adjust the volume and pH. Finally, measure the enzyme activity in a fluorescence reader equipped with the appropriate filter set, and analyze the data using the appropriate software for the fluorescence reader device.
