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Methods Cell Biol. 2014;122:193-218. doi: 10.1016/B978-0-12-417160-2.00009-6.

Xenopus in vitro assays to analyze the function of transmembrane nucleoporins and targeting of inner nuclear membrane proteins.

Author information

1
Friedrich Miescher Laboratory of the Max Planck Society, Tübingen, Germany.

Abstract

Xenopus egg extracts have been widely used to study cell cycle regulation and to analyze mitotic or nuclear processes on a biochemical level. Most instrumental, proteins of interest can be immunodepleted by specific antibodies. However, this approach has been restricted to non-membrane proteins, which limits its versatility especially when studying membrane-dependent processes such as nuclear envelope reformation at the end of mitosis or nuclear pore complex assembly. We describe here the methods developed and used in our laboratory to specifically remove transmembrane proteins from endogenous membranes and to insert recombinant integral membrane proteins into endogenous membranes. The latter procedure is important not only for readdition of a depleted protein in rescue experiments but also for introducing artificial membrane proteins such as reporters to investigate the passage of inner nuclear membrane proteins through nuclear pore complexes.

KEYWORDS:

Cell-free assay; Immunodepletion; Integral membrane protein; Membrane reconstitution; Nuclear envelope; Nuclear pore complex; Nuclear reformation; Nuclear transport; Proteoliposomes; Xenopus egg extracts

[Indexed for MEDLINE]

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