Analysis of Ataxin-2 interactome using LC-MS/MS

We analyzed Ataxin-2 interactome using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). HEK293T cell lysate was subjected to immunoprecipitation (IP) with either anti-Ataxin-2 antibody or control IgG. The interaction between Ataxin-2 and the copurified proteins do not appear to be mediated by RNA, as the immunoprecipitation experiments were performed under buffer conditions that include RNase A and RNase I. The proteins purified using anti-Ataxin-2 and control IgG were separated by SDS-PAGE. CBB-stained gel was divided into three parts per lane in order to increase the detection sensitivity. Proteins extracted from the gels were analyzed by liquid chromatography coupled to tandem mass spectrometry to identify their protein content.