C myc tag ip co ip kit

To extract nuclear proteins, purified nuclei (100 μg protein) were suspended in 60 μl of suspension buffer [25 mM sucrose, 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 10 mM CaCl₂] and mixed with 60 μl of 0.4 M KCl containing 1 mM PMSF and protease inhibitors (final concentrations of 2 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A). The suspension was gently rocked for 2 hr at 4°. Insoluble material was removed by centrifugation at 13,000 rpm (16,060 × g) for 30 min at 4° in a Sorvall Biofuge fresco centrifuge. The supernatant containing salt-extracted proteins was loaded onto a desalting column (Zeba Spin Desalting column; Thermo Scientific, Rockford, IL) which was placed into a fresh 1.5 ml Eppendorf tube. The desalting column was centrifuged at 1500 × g (4000 rpm) for 2 min at 4° in a Sorvall Biofuge fresco centrifuge. The flow-through was immediately used in coimmunoprecipitation experiments with the Pierce ProFoundTM HA or c-Myc Tag IP/Co-IP kit (Thermo Scientific).