Table 1.

The fluorometric plate assay for ARSB uses cell homogenate, not cell lysate. Units are expressed as nmol/mg protein/h [39,54].

Method for Measurement of ARSB Activity by Fluorometric Microplate Assay with 4-Methylumbelliferyl Sulfate
1	The substrate is 5 mM of 4-methylumbelliferyl sulfate (4-MUS) in assay buffer, made fresh.
2	The assay buffer is 0.05 M Na acetate with 20 mM barium acetate, pH 5.6, at 37 °C.
3	Cell homogenate is prepared in ddH2O on ice, by sonication with metal tip, three times for 10 s.
4	20 µL cell homogenate is combined with 80 μL assay buffer and 100 μL substrate in wells of a microplate.
5	The microplate is incubated for 30 min at 37 °C.
6	The reaction is stopped by 150 μL of stop buffer (glycine 350 mM–carbonate 440 mM, pH 10.7).
7	Fluorescence is measured at 360 nm (excitation) and 465 nm (emission).
8	Enzymatic activity is expressed as nmol/mg protein/h using the protein content of the cell extract and a standard curve of 4-methylumbelliferone (MU) of known concentration.
Reagents	4-Methylumbelliferyl sulfate potassium salt (4-MUS; MW 294.32; C10H7KO6S); Na-acetate (MW 82.03; CH3COONa); Glycine (MW 75.07; NH2CH2COOH); Carbonate (MW 124.00; Na2CO3); Barium acetate [MW 255.42; (CH3COO)2Ba]
Assay Buffer	Na-acetate buffer 0.05 M with barium acetate 20 mM; pH 5.6
Substrate	5 mM 4-MUS substrate in Na-acetate/barium acetate buffer (fresh)
Glycine-carbonate stop buffer	Glycine 350 mM/Carbonate 440 mM; pH 10.7