Download Hoefer DQ 200 - Genemco, Inc.

5. Enzyme Activity Quantitation
Esters of 4-methylumbelliferone (4MU, 7-hydroxy4-methylcoumarin) do not fluoresce unless cleaved
to release the free fluorophore. Free 4MU can
be used as a standard to calibrate fluorometric
enzyme assays based on the hydrolysis of 4MUcontaining substrates such as β-4-MU-glucuronide
by β-glucuronidase (GUS), or β-4-MU-galactose
by β-galactosidase. A solution of 4MU can also
be used to check instrument performance. The
accuracy of this assay is also dependent on the
accuracy of the start and stop times for enzyme
digestion of the substrate.
Specific protocols for assaying β-galactosidase
and β-glucuronidase activities are in Appendix A.
Solutions
4MU stock solution A
1 mM (100 ml)
4-methylumbelliferone (sodium salt),
(MW = 198.20)
19.8 mg
Distilled water
to 100 ml
Store at 4 °C, away from light.
4MU stock solution B
1 µM 4MU (10 ml)
4MU stock solution A
10 µl
Distilled water
10 ml
Store at 4 °C, away from light.
Carbonate stop buffer
• p26
0.20 M (1000 ml)
Sodium carbonate, anhydrous,
(MW=105.99)
21.2 g
Double distilled water
to 1000 ml