Automodification activity of PARP1 was assayed as previously described23 (link). Briefly, PARP1 (#4668, Trevigen) was incubated for 20 minutes at room-temperature in reaction buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM MgCl2) containing 130 ng of activated DNA (#4671-096-06, Trevigen) in the presence or absence of 200 µM NAD (#4684-096-02, Trevigen) and in the presence or absence of 0.1 M H2SO4. Reactions were stopped with 2 µM Olaparib and analyzed by Western blotting.
Protocol detail
Quantification of PARP1 Automodification
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Other organizations : Max Planck Institute for Biology of Ageing, University of Oxford
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Variable analysis
independent variables
- Presence or absence of 200 µM NAD
- Presence or absence of 0.1 M H2SO4
- Automodification activity of PARP1
- PARP1 (#4668, Trevigen)
- Reaction buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM MgCl2)
- Activated DNA (#4671-096-06, Trevigen)
- Incubation time (20 minutes)
- Temperature (room-temperature)
- Positive control: Presence of 200 µM NAD
- Negative control: Absence of 200 µM NAD
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