Improving ChIP Efficiency by Removing Soluble CSB

To increase ChIP efficiency we removed soluble CSB before cross-linking DNA and proteins [8] (link), [35] (link), [58] (link). Cells were collected in Buffer B (150 mM NaCl, 0.5 mM MgCl₂, 20 mM HEPES pH 7.8, 10% Glycerol, 0.5% Triton X-100) and soluble CSB was separated from chromatin by centrifugation at 15,000 RPM for 5 min at 4°C. The resulting pellets were resuspended in Buffer B and fixed with 1% formaldehyde for 10 min at room temperature. Cross-linked cells were sonicated at 40% amplitude (30 sec on, 90 sec off, for 24 min total) using the Branson 101-135-126 Sonifier. Chromatin IP (ChIP) was performed using a monoclonal anti-CSB antibody (1B1) that recognizes the N-terminal 507 amino acids of CSB [35] (link), [39] (link) or an anti-c-Jun antibody (Santa Cruz, sc-1694). ChIP samples were reverse cross-linked in SDS sample buffer for subsequent western blot analyses [20] (link). Antibodies used for western blot analysis are rabbit anti-CSB antibodies (kindly provided by Dr. Alan Weiner, U. Washington) [35] (link), c-Jun (Santa Cruz, sc-1694) and GAPDH (Millipore, MAB374).