Table 1.

SMO enzyme activity assay setup and workflow

Reagent	Stock concentration	Final concentration	Per reaction (μL)	Per 100 reactions
Glycine, pH 8.0	0.5 M	0.083 M	50	5 mL
Aminoguanidine	100 mM	1 mM	3	300 μL
Pargyline	2.3 mg/mL	0.019 mg/mL	2.5	250 μL
Luminol	100 mM	0.017 mM	0.05	5 μL
HPLC-grade water			94.5	9.45 mL
Horseradish peroxidase	0.4 mg/mL	0.067 mg/mL	50	5 mL
Reaction mix			200
Cell lysate or 0.083 M glycine blank			50
Vortex cuvette briefly and incubate for 2 min in 37 °C water bath
Spermine	1.5 mM	0.25 mM	50
Final reaction volume			300
Inject substrate and integrate luminescence for 40 s

The necessary assay reagents are indicated above as well as calculations for preparing a master mix for 100 reactions (see Subheading 2 for preparation instructions). The general assay scheme is also summarized (see Subheading 3 for detailed protocol)