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Cold Spring Harb Protoc. 2018 Sep 4;2018(9). doi: 10.1101/pdb.prot097113.

Protein Immunodepletion and Complementation in Xenopus laevis Egg Extracts.

Author information

1
Laboratory of Chromosome and Cell Biology, The Rockefeller University, New York, New York 10065.
2
Department of Biology, The College of New Jersey, Ewing, New Jersey 08628.
3
Laboratory of Chromosome and Cell Biology, The Rockefeller University, New York, New York 10065; funabih@rockefeller.edu.

Abstract

The Xenopus egg extract system has been widely used to study cell cycle events, including DNA replication, nuclear envelope formation, spindle assembly, chromosome condensation and kinetochore formation. The functional roles of the proteins involved in these processes can be determined by immunodepleting a protein of interest from the extract. As immunodepletion may result in co-depletion of other proteins, the protein of interest can be added back to the extract to verify its function. Additionally, proteins harboring point mutations or domain deletions may be added to assess their functions. Here we outline the immunodepletion procedure and two separate methods for restoring a protein of interest: addition of either a recombinant protein or an mRNA that supports translation in egg extracts. The tradeoffs between these two methods are discussed.

PMID:
29382808
DOI:
10.1101/pdb.prot097113

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