Nuclear Magnetic Resonance:Article Title: PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2B Glu2
Article Snippet: .. For NMR experiments, the protein was expressed in 3 l filter-sterilized Overnight Express Autoinduction NMR Media (Merck-Millipore, Billerica, MA, USA) containing 50 mM [ 15 N] NH 4 Cl and 0.5% (w/v) [ 13 C 3 ] glycerol (CortecNet, Voisins-le-Bretonneux, France) at a temperature of 25 °C for 24 h. The resulting cell pellets were resuspended in 50 ml Buffer A (50 mM HEPES-HCl pH 7.5, 0.5 M NaCl, 0.5 mM TCEP) supplemented with protease inhibitors (Roche, Burgess Hill, UK), lysed by sonication, and the clarified supernatant applied to a 5 ml Talon (TaKaRa Bio, Saint-Germain-en-Laye, France) affinity column by gravity flow in Buffer A. .. After successive washes in Buffer A (20 CV), bound protein was eluted in Buffer B (50 mM HEPES-HCl pH 7.4, 0.5 M NaCl, 0.5 mM TCEP, 0.3 M imidazole) and eluted cPARP3 concentrated using Vivaspin 20 (30,000 MWCO) centrifugal concentrators (Sartorius Stedim Biotech, Goettingen, Germany). cPARP3 was diluted fivefold in Buffer C (50 mM HEPES-HCl pH 7.5, 150 mM NaCl, 0.5 mM TCEP) and further purified on a 5 ml HiTrap Heparin HP column (GE Healthcare, Little Chalfont, UK) using a linear salt gradient.
Article Title: Efficient Single-Strand Break Repair Requires Binding to Both Poly(ADP-Ribose) and DNA by the Central BRCT Domain of XRCC1
Article Snippet: .. For NMR experiments, the protein was expressed in 1 l filter-sterilized Overnight Express Autoinduction NMR Media (Merck-Millipore, Billerica, MA, USA) containing 50 mM [ 15 N] NH4Cl and 0.5% (w/v) [ 13 C3] glycerol (CortecNet, Voisins-le-Bretonneux, France) at a temperature of 25°C for 30 h. The cell pellet resulting from 4 l of culture was resuspended in Buffer A (50 mM HEPES.NaOH pH 7.5, 250 mM NaCl, 10 mM imidazole, 0.5 mM TCEP) supplemented with protease inhibitor tablets (Roche, Burgess Hill, UK). ..
Sonication:Article Title: PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2B Glu2
Article Snippet: .. For NMR experiments, the protein was expressed in 3 l filter-sterilized Overnight Express Autoinduction NMR Media (Merck-Millipore, Billerica, MA, USA) containing 50 mM [ 15 N] NH 4 Cl and 0.5% (w/v) [ 13 C 3 ] glycerol (CortecNet, Voisins-le-Bretonneux, France) at a temperature of 25 °C for 24 h. The resulting cell pellets were resuspended in 50 ml Buffer A (50 mM HEPES-HCl pH 7.5, 0.5 M NaCl, 0.5 mM TCEP) supplemented with protease inhibitors (Roche, Burgess Hill, UK), lysed by sonication, and the clarified supernatant applied to a 5 ml Talon (TaKaRa Bio, Saint-Germain-en-Laye, France) affinity column by gravity flow in Buffer A. .. After successive washes in Buffer A (20 CV), bound protein was eluted in Buffer B (50 mM HEPES-HCl pH 7.4, 0.5 M NaCl, 0.5 mM TCEP, 0.3 M imidazole) and eluted cPARP3 concentrated using Vivaspin 20 (30,000 MWCO) centrifugal concentrators (Sartorius Stedim Biotech, Goettingen, Germany). cPARP3 was diluted fivefold in Buffer C (50 mM HEPES-HCl pH 7.5, 150 mM NaCl, 0.5 mM TCEP) and further purified on a 5 ml HiTrap Heparin HP column (GE Healthcare, Little Chalfont, UK) using a linear salt gradient.
Affinity Column:Article Title: PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2B Glu2
Article Snippet: .. For NMR experiments, the protein was expressed in 3 l filter-sterilized Overnight Express Autoinduction NMR Media (Merck-Millipore, Billerica, MA, USA) containing 50 mM [ 15 N] NH 4 Cl and 0.5% (w/v) [ 13 C 3 ] glycerol (CortecNet, Voisins-le-Bretonneux, France) at a temperature of 25 °C for 24 h. The resulting cell pellets were resuspended in 50 ml Buffer A (50 mM HEPES-HCl pH 7.5, 0.5 M NaCl, 0.5 mM TCEP) supplemented with protease inhibitors (Roche, Burgess Hill, UK), lysed by sonication, and the clarified supernatant applied to a 5 ml Talon (TaKaRa Bio, Saint-Germain-en-Laye, France) affinity column by gravity flow in Buffer A. .. After successive washes in Buffer A (20 CV), bound protein was eluted in Buffer B (50 mM HEPES-HCl pH 7.4, 0.5 M NaCl, 0.5 mM TCEP, 0.3 M imidazole) and eluted cPARP3 concentrated using Vivaspin 20 (30,000 MWCO) centrifugal concentrators (Sartorius Stedim Biotech, Goettingen, Germany). cPARP3 was diluted fivefold in Buffer C (50 mM HEPES-HCl pH 7.5, 150 mM NaCl, 0.5 mM TCEP) and further purified on a 5 ml HiTrap Heparin HP column (GE Healthcare, Little Chalfont, UK) using a linear salt gradient.
Protease Inhibitor:Article Title: Efficient Single-Strand Break Repair Requires Binding to Both Poly(ADP-Ribose) and DNA by the Central BRCT Domain of XRCC1
Article Snippet: .. For NMR experiments, the protein was expressed in 1 l filter-sterilized Overnight Express Autoinduction NMR Media (Merck-Millipore, Billerica, MA, USA) containing 50 mM [ 15 N] NH4Cl and 0.5% (w/v) [ 13 C3] glycerol (CortecNet, Voisins-le-Bretonneux, France) at a temperature of 25°C for 30 h. The cell pellet resulting from 4 l of culture was resuspended in Buffer A (50 mM HEPES.NaOH pH 7.5, 250 mM NaCl, 10 mM imidazole, 0.5 mM TCEP) supplemented with protease inhibitor tablets (Roche, Burgess Hill, UK). ..
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