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

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

Methods in molecular biology (Clifton, N.J.) (2016-05-06)
Nadia Korfali, Laurence Florens, Eric C Schirmer
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Benzonase® Nuclease, ultrapure, ≥250 units/μL, ≥99% (SDS-PAGE), recombinant, expressed in E. coli, buffered aqueous glycerol solution, ultrapure grade
Sigma-Aldrich
Deoxyribonuclease I from bovine pancreas, Type II, lyophilized powder, Protein ≥80 %, ≥2,000 units/mg protein
Sigma-Aldrich
Anti-LAP2 Antibody, from rabbit, purified by affinity chromatography