Anti rabbit igg 2729

Cells were fixed with 1% formaldehyde for 15 min in room temperature and quenched with 0.125 M glycine for 5 minutes in room temperature. ChIP assays was performed using a commercially available kit (ChIP-IT Express, Active Motif). Briefly, cell pellet was lysed to isolate chromatin using lysis buffer, followed by disruption with a Dounce homogenizer. LAM 621–101 cells chromatin was sheared enzymatically for 10 minutes at 37 C (ChIP-IT Express Enzymatic° Shearing Kit, Active Motif). Immunoprecipitation was performed using anti-cMyc (9402, Cell Signaling) or anti-Rabbit IgG (2729, Cell Signaling) antibodies. Complexes were washed, eluted from the beads with elution buffer containing SDS buffer, and followed by RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65°C, and ChIP DNA was purified with a Chromatin IP DNA purification kit (Active Motif) and qPCR was performed with SYBR Green master mix on a Step One Real-Time PCR system (all Life Technologies) using the primer sets for MAP2K1, CCNB1, CDK4, RPL4, and RPL35 listed in Table S3 (Barrilleaux et al., 2013 (link); Ciribilli and Borlak, 2017 ; Menssen and Hermeking, 2002 (link)).