Affinity purification of ADPr proteins

Cells were pelleted, washed with cold KK2, and resuspended in lysis buffer (50 mM Tris HCl pH 8.0, 10 mM NaCl, 3 mM MgCl₂, 3 mM CaCl₂, 0.5 M Sorbitol, 0.6% Triton X-100; with protease inhibitors (Complete cocktail-Roche^®), PARP (50 μM Benzamide) and PARG (200 μM DEA) inhibitors) at a concentration of 100 × 10⁶ cells/ml, incubated with rotation at 4 °C for 10 min, and the nuclei pelleted by centrifugation at 2300 g for 5 min. Extracted nuclei were resuspended/washed with the same buffer. 10% of the nuclei were collected as the Input. The rest of the nuclei were lysed with GnHCl buffer (6 M Guanidine hydrochloride, 50 mM TrisPh8.5) for 30 min with rotation at 4 °C. Then, the samples were diluted 10 times in IP buffer (50 mM Tris HCl pH8, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 1% Triton X-100 + inhibitors). 20 µl of prior washed Dynabead^®-Protein G (Invitrogen^®) was added for 1 h. Meanwhile, 3 µg of PAN-ADPr reagent (Merk^® MABE1016) was incubated with 30 µl of Dynabead^®-Protein G (Invitrogen^®) for 1 h in 0.01% Tween-20 in KK2. Pre-cleared samples were incubated with the conjugated beads at 4 °C on a rotating wheel. The beads were washed for 5 min, with the following buffers: W1 (Tris pH8 10 mM, KCl 150 mM, NP40 0.5%, EDTA 1 mM), W2 (Tris pH8 10 mM, NaCl 200 mM, TritonX100 0.5%) W3 (Tris pH8 10 mM, NaCl 400 mM, TritonX100 0.5%), W4 (Tris pH8 10 mM, NaCl 500 mM, TritonX100 0.5%), W5 (Tris pH8 10 mM, LiCl 250 mM, NP40 0.5%, EDTA 1 mM) and W6 (Tris pH8 10 mM, EDTA 1 mM). Elution was performed by boiling samples in SDS buffer. Samples were analysed by immunoblotting.