Purification of Lamins and Soluble Fragments of NETs

Methods in Enzymology
Alexandr A MakarovEric C Schirmer

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 cons...Continue Reading

Citations

Oct 3, 2019·Nature Cell Biology·Adam KaroutasAsifa Akhtar
Jul 13, 2019·Nature Communications·Alex A MakarovEric C Schirmer
Feb 20, 2019·Cells·Marianne GrafeRalph Gräf
Jan 22, 2021·Cytoskeleton·Ecem Kural-Mangıt, Pervin Rukiye Dinçer

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