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Isolation, Proteomic Analysis, and Microscopy Confirmation of the Liver Nuclear Envelope Proteome

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Original languageEnglish
Title of host publicationThe Nuclear Envelope
Subtitle of host publicationMethods and Protocols, Methods in Molecular Biology
EditorsSue Shackleton
Place of PublicationNew York
PublisherSpringer Science + Business Media
Pages3-44
Number of pages42
Volume1411
DOIs
StatePublished - 2016

Publication series

NameMethods in molecular biology (Clifton, N.J.)
PublisherHumana Press
ISSN (Print)1064-3745

Abstract

Nuclei can be relatively easily extracted from homogenized liver due to the softness of the tissue and crudely separated from other cellular organelles by low-speed centrifugation due to the comparatively large size of nuclei. However, further purification is complicated by nuclear envelope continuity with the endoplasmic reticulum, invaginations containing mitochondria, and connections to the cytoskeleton. Subsequent purification to nuclear envelopes is additionally confounded by connections of inner nuclear membrane proteins to chromatin. For these reasons, it is necessary to confirm proteomic identification of nuclear envelope proteins by testing targeting of individual proteins. The proteomic identification of nuclear envelope fractions is affected by the tendencies of transmembrane proteins to have extreme isoelectric points, strongly hydrophobic peptides, posttranslational modifications, and a propensity to aggregate, thus making proteolysis inefficient. To circumvent these problems, we have developed a MudPIT approach that uses multiple extractions and sequential proteolysis to increase identifications. Here we describe methods for isolating nuclear envelopes, determining their proteome by MudPIT, and confirming their targeting to the nuclear periphery by microscopy.

    Research areas

  • Animals, Capillary Electrochromatography, Chemical Fractionation, Chromatography, Liquid, Computational Biology, Liver/metabolism, Membrane Proteins/isolation & purification, Microscopy, Fluorescence, Nuclear Envelope/metabolism, Protein Transport, Proteome, Proteomics/methods, Rats, Tandem Mass Spectrometry

ID: 82533149