The EnzChek Pyrophosphate
Assay kit was purchased from Thermo Fisher. Each reaction contains
the following components: 100 mM Tris pH 8.0, 50 mM NaCl, 10 mM MgCl2, 5 mM DTT, 200 μM 2-amino-6-mercapto-7-methylpurine
riboside (MESG), 1 U purine nucleoside phosphorylase (PNP), and when
indicated 10 μM Mycobacterium tuberculosis pyrophosphatase
(MtPPase, a kind gift from Dr. Luiz Pedro de Carvalho
of the Francis Crick Institute). Concentrations of enzymes, peptides,
and nucleotides were varied in individual reactions. MtPPase was purified following an established protocol.20 (link) Assays were carried out in 100 μL or 200
μL reaction volumes in 96-well plates, and the absorbance at
360 nm was monitored by a SpectraMax plate reader (Molecular Devices).
Assay kit was purchased from Thermo Fisher. Each reaction contains
the following components: 100 mM Tris pH 8.0, 50 mM NaCl, 10 mM MgCl2, 5 mM DTT, 200 μM 2-amino-6-mercapto-7-methylpurine
riboside (MESG), 1 U purine nucleoside phosphorylase (PNP), and when
indicated 10 μM Mycobacterium tuberculosis pyrophosphatase
(MtPPase, a kind gift from Dr. Luiz Pedro de Carvalho
of the Francis Crick Institute). Concentrations of enzymes, peptides,
and nucleotides were varied in individual reactions. MtPPase was purified following an established protocol.20 (link) Assays were carried out in 100 μL or 200
μL reaction volumes in 96-well plates, and the absorbance at
360 nm was monitored by a SpectraMax plate reader (Molecular Devices).
