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Xenopus in vitro assays to analyze the function of transmembrane nucleoporins and targeting of inner nuclear membrane proteins.
- Source :
-
Methods in cell biology [Methods Cell Biol] 2014; Vol. 122, pp. 193-218. - Publication Year :
- 2014
-
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.<br /> (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Subjects :
- Animals
Antibodies, Monoclonal immunology
Escherichia coli genetics
Escherichia coli metabolism
Gene Expression
Membrane Fusion
Membrane Proteins analysis
Membrane Proteins biosynthesis
Microscopy, Electron, Transmission
Nuclear Pore metabolism
Ovum cytology
Protein Transport
Recombinant Proteins genetics
Xenopus laevis
Membrane Proteins metabolism
Nuclear Envelope metabolism
Nuclear Pore Complex Proteins analysis
Ovum metabolism
Recombinant Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0091-679X
- Volume :
- 122
- Database :
- MEDLINE
- Journal :
- Methods in cell biology
- Publication Type :
- Academic Journal
- Accession number :
- 24857731
- Full Text :
- https://doi.org/10.1016/B978-0-12-417160-2.00009-6