Protocol detail

TARG1-mediated PAR hydrolysis assay

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TARG1-type macrodomain PAR hydrolysis was determined by western blot^{33 (link)}. The substrate used was automodified hPARP obtained as described above using NAD⁺ and activated DNA as substrates and Rucaparib to stop the reaction. Then, this PARylated substrate was incubated for 1 hour at 30 °C in the presence of FmTARG1 (20 μM) or hTARG1 (10 μM), before running the samples on 7–10% SDS-PAGE gels and transfering to a nitrocellulose membrane. The presence of PAR was detected by coupling with rabbit polyclonal anti-PAR antibodies (1:1000, Trevigen) and goat-anti-rabbit-HRP conjugated secondary antibody (Bio-Rad), and finally revealed with Opti-4CN substrate (Bio-Rad) for 5 minutes. Analysis of the reaction products of hTARG1 and FmTARG1 on poly (ADP-ribosyl)ated PARP1 was carried out by mass spectrometry as described above for the de-MARylation assay.

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García-Saura A.G., Zapata-Pérez R., Hidalgo J.F., Cabanes J., Gil-Ortiz F, & Sánchez-Ferrer Á. (2019). An uncharacterized FMAG_01619 protein from Fusobacterium mortiferum ATCC 9817 demonstrates that some bacterial macrodomains can also act as poly-ADP-ribosylhydrolases. Scientific Reports, 9, 3230.

Publication 2019

anti-PAR antibodies goat-anti-rabbit-HRP conjugated secondary antibody Opti-4CN substrate

Anti antibodies Antibody Assay Gels Goat Hydrolysis Marylation Mass spectrometry Membrane Nitrocellulose Parp1 Poly Rabbit Rucaparib Sds page

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Variable analysis

independent variables

FmTARG1 (20 μM)
HTARG1 (10 μM)

dependent variables

PAR hydrolysis as determined by western blot

control variables

Automodified hPARP obtained using NAD+ and activated DNA as substrates
Rucaparib to stop the reaction
Incubation at 30 °C for 1 hour
7–10% SDS-PAGE gels and transfer to nitrocellulose membrane
Rabbit polyclonal anti-PAR antibodies (1:1000, Trevigen) and goat-anti-rabbit-HRP conjugated secondary antibody (Bio-Rad)
Opti-4CN substrate (Bio-Rad) for 5 minutes

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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