Work

Use of Sequential Chemical Extractions to Purify Nuclear Membrane Proteins for Proteomics Identification

Year: 2009

Type: book-chapter

Abstract: The nuclear envelope (NE) is a double membrane system that is both a part of the endoplasmic reticulum and part of the nucleus. As its constituent proteins tend to be highly complexed with nuclear and cytoplasmic components, it is notoriously difficult to purify. Two methods can reduce this difficulty for the identification of nuclear membrane proteins: comparison to contaminating membranes and chemical extractions to enrich for certain groups of proteins. The purification of nuclear envelopes and contaminating microsomal membranes is described here along with procedures for chemical extraction using salt and detergent, chaotropes, or alkaline solutions. Each extraction method enriches for different combinations of nuclear envelope proteins. Finally, we describe the analysis of these fractions with MudPIT, a proteomics methodology that avoids gel extraction of bands to facilitate identification of minor proteins and membrane proteins that do not resolve well on gels. Together these three approaches can significantly increase the output of proteomics studies aimed at identifying the protein complement of subcellular membrane systems.

Source: Methods in molecular biology

DOI: 10.1007/978-1-60327-310-7_15

Authors: Nadia Korfali, Elizabeth A.L. Fairley, Selene K. Swanson, Laurence Florens, Eric C. Schirmer

Institutions: Wellcome Centre for Cell Biology, University of Edinburgh, Stowers Institute for Medical Research

Language: English

Topic: Advanced Proteomics Techniques and Applications

Subfield: Spectroscopy

Field: Chemistry

Domain: Physical Sciences

Open Access status: green

Funder: Wellcome Trust

Award: The role of the nuclear envelope in genome architecture and cellular differentiation.