title: Simple Separation of Functionally Distinct Populations of Lamin-Binding Proteins.
abstract: The inner membrane of the nuclear envelope (NE) is home to hundreds of integral membrane proteins (NE transmembrane proteins, "NETs") with conserved or tissue-specific roles in genome organization and nuclear function. Nearly all characterized NETs bind A- or B-type lamins directly. However, hundreds of NETs remain uncharacterized, collectively posing an enormous gap that must be bridged to understand nuclear function and genome biology. We provide technically simple protocols for the separation and recovery of functionally distinct populations of NETs and A-type lamins. This protocol was developed for emerin, an inner nuclear membrane protein that binds lamins and barrier-to-autointegration factor (BANF1) as a component of nuclear lamina structure, and has diverse roles in nuclear assembly, signaling, and gene regulation. This protocol separates easily solubilized ("easy") populations of nuclear lamina proteins (emerin, lamin A, BAF) from "sonication-dependent" populations. Depending on cell type, the "easy" and "sonication-dependent" fractions each contain up to about half the available emerin, A-type lamins, and BAF, whereas B-type lamins and histone H3 are predominantly sonication dependent. The two populations of emerin have distinct posttranslational modifications, and only one population associates with BAF. This method may be useful for functional screening or analysis of other lamin-associated proteins, including novel NETs emerging from proteomic studies.
authors.affiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Berk
authors.affiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Electronic address: klwilson@jhmi.edu.
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Wilson
mesh_terms: D002458:Cell Fractionation; D002498:Centrifugation; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D028861:Nuclear Pore Complex Proteins / Q000302:isolation & purification*
chemical_list: D028861:Nuclear Pore Complex Proteins
keywords: Emerin; Fractionation; LEM domain; Lamin; Laminopathy; Nuclear envelope; Nuclear isolation; Nuclear lamina; Nucleoskeleton