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Methods Mol Biol. 2016;1400:311-38. doi: 10.1007/978-1-4939-3372-3_20.

Characterization of L1-Ribonucleoprotein Particles.

Author information

1
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
2
Department of Pathology, Massachusetts General Hospital, Baltimore, MA, 21205, USA.
3
Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY, 10021, USA.
4
Department of Biochemistry and Molecular Pharmacology and Institute for Systems Genetics, New York University Langone School of Medicine, 550 First Avenue, New York, NY, 10016, USA.
5
Department of Molecular Biology and Genetics, High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
6
Modern Meadow, Inc., Brooklyn, NY, 11220, USA.
7
Department of Biochemistry and Molecular Pharmacology and Institute for Systems Genetics, New York University Langone School of Medicine, 550 First Avenue, New York, NY, 10016, USA. Jef.Boeke@nyumc.org.

Abstract

The LINE-1 retrotransposon (L1) encodes two proteins, ORF1p and ORF2p, which bind to the L1 RNA in cis, forming a ribonucleoprotein (RNP) complex that is critical for retrotransposition. Interactions with both permissive and repressive host factors pervade every step of the L1 life cycle. Until recently, limitations in detection and production precluded in-depth characterization of L1 RNPs. Inducible expression and recombinant engineering of epitope tags have made detection of both L1 ORFs routine. Here, we describe large-scale production of L1-expressing HEK-293T cells in suspension cell culture, cryomilling and affinity capture of L1 RNP complexes, sample preparation for analysis by mass spectrometry, and assay using the L1 element amplification protocol (LEAP) and qRT-PCR.

KEYWORDS:

Affinity purification; Cryomilling; Interactomics; LINE-1; Mass spectrometry; Metabolic labeling; Protein complexes; Ribonucleoprotein

PMID:
26895062
PMCID:
PMC5084692
DOI:
10.1007/978-1-4939-3372-3_20
[Indexed for MEDLINE]
Free PMC Article

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