Ab110328

Anti-FXN antibody ab110328 (abcam) was utilized for immunoprecipitation of FXN protein from lysates. To cross-link antibodies to protein A/G beads (Santa Cruz Biotech), 500 μl resin was washed in PBS and then incubated with 100 μl ab110328 antibody for 1 h at room temperature with end-over-end rotation. Beads were then washed with PBS and covalent conjugation of antibody was carried out using a freshly prepared primary amine cross-linking buffer (180 μl 2.5 mM DSS from Pierce, 770 μl water, and 50 μl 20 × PBS). Residual DSS and unbound antibody were removed by washing with 0.1 M Glycine pH 2.5–3 followed by washes in PBS. Finally, beads were stored at 4 °C in PBS. For immunoprecipitation (IP), 500 μg of cleared cell lysates (containing NEM) were incubated with 25 μl of anti-FXN conjugated beads for 2 h at 4 °C then washed two times with lysis buffer (Fig. S3B) or in high (500 mM NaCl, 50 mM TrisCl pH 7.5, 0.5% Igepal CA-630) and low salt buffers (10 mM TrisCl, 0.5% Igepal CA-630; Fig. S3C) before removal of trace amounts of lysis buffer. Resultant IP beads were resuspended in 50 μl 2x LDS sample buffer and heated at 100 °C for 5 min. Eluates were then carefully transferred, without beads, to clean microtubes and supplemented with DTT reducing agent. Input lysates, IPs, and output flowthrough lysates were analyzed by immunoblotting as described above.