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Updated: Aug 27, 2025
Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale
Published on: May 17, 2014
Mass spectrometry analysis of affinity-purified cytoplasmic translation initiation complexes from human and fly cells
Chingakham Ranjit Singh1, Naoki Tani2, Akira Nakamura2
1Molecular Cellular and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, KS 66506, USA; Department of Pediatrics, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Abstract:
eIF5-mimic protein (5MP) controls translation through binding to the ribosomal pre-initiation complex (PIC) and alters non-AUG translation rates for cancer oncogenes and repeat-expansions in neurodegenerative diseases. Here, we describe a semi-quantitative protocol for detecting 5MP-associated proteins in cultured human and fly cells. We detail one-step anti-FLAG affinity purification and whole-lane mass spectrometry analysis of samples resolved by SDS-PAGE. This protocol allows for quantitative evaluation of the effect of 5MP mutations on its molecular interactions, to elucidate translational control by 5MP. For complete details on the use and execution of this protocol, please refer to Singh et al. (2021).
