Rabbit anti igg

HeLa cells were grown in 100 mm dishes until 95% - 100% confluent. Following stress, cells were crosslinked with 0.1 % formaldehyde and quenched with 1.25 M glycine. Cells were subsequently scraped into IP lysis buffer (50 mM HEPES pH 7.2, 150 mM NaCl, 0.1 % NP-40, 10% Glycerol, 2 mM EDTA, 2 mM EGTA, 1 mM DTT, Protease inhibitor (Sigma Aldrich) and Ribolock (ThermoFisher)), and incubated on a rotator at 4°C for 20 mins. Lysates were centrifuged at 10,000 × g for 15 mins at 4°C and the supernatant was collected. Protein content of the supernatants was determined using the Bradford protein assay (BioRad). Each immunoprecipitation was carried out in quadruplicate. 1 μg of rabbit anti-Caprin-1 (Proteintech) or rabbit anti-IgG (Jackson Immuno Research Laboratories) was added to magnetic protein A Dynabeads (ThermoFisher). Immunoprecipitation was performed with an input of 400 μg of total protein for 1 hr at room temperature. After IP, the beads were washed 6 times with IP lysis buffer (without DTT, Protease inhibitors, and Ribolock) and stored at −20°C.