Purification of Lamins and Soluble Fragments of NETs

Alexandr A. Makarov, Andrea Rizzotto, Peter Meinke, Eric C. Schirmer

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Lamins and associated nuclear envelope transmembrane proteins (NETs) present unique problems for biochemical studies. Lamins form insoluble intermediate filament networks, associate with chromatin, and are also connected via specific NETs to the cytoskeleton, thus further complicating their isolation and purification from mammalian cells. Adding to this complexity, NETs at the inner nuclear membrane function in three distinct environments: (a) their nucleoplasmic domain(s) can bind lamins, chromatin, and transcriptional regulators; (b) they possess one or more integral transmembrane domains; and (c) their lumenal domain(s) function in the unique reducing environment of the nuclear envelope/ER lumen. This chapter describes strategic considerations and protocols to facilitate biochemical studies of lamins and NET proteins in vitro. Studying these proteins in vitro typically involves first expressing specific polypeptide fragments in bacteria and optimizing conditions to purify each fragment. We describe parameters for choosing specific fragments and designing purification strategies and provide detailed purification protocols. Biochemical studies can provide fundamental knowledge including binding strengths and the molecular consequences of disease-causing mutations that will be essential to understand nuclear envelope-genome interactions and nuclear envelope linked disease mechanisms.

Original languageEnglish
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages79-100
Number of pages22
Volume569
ISBN (Print)9780128034699
DOIs
Publication statusPublished - 22 Jan 2016

Publication series

NameMethods in Enzymology
Volume569
ISSN (Print)00766879
ISSN (Electronic)15577988

Keywords

  • Binding assay
  • Intermediate filaments
  • Lamin
  • Nuclear envelope
  • Nuclear envelope transmembrane protein

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