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Xenopus egg extract represents a powerful cell-free biochemical tool for studying organelle assembly and function. Large quantities of cytoplasm can be isolated, and biochemical manipulation of extract composition and cell cycle state is relatively straightforward. In this protocol, we describe the reconstitution of nuclear assembly by adding a chromatin source to interphasic X. laevis egg extract. Intact nuclei assemble within 30-45 min of initiating the reaction, followed by nuclear growth. We also describe methods for imaging and quantifying nuclear import kinetics. Recombinant proteins or small molecules of interest can be added to the extract before or after nuclear assembly, and immunodepletion allows for removal of specific proteins from the extract. This approach will continue to inform mechanisms of nuclear assembly, nuclear pore complex assembly and function, nucleocytoplasmic transport, DNA replication, nuclear envelope breakdown, and nuclear size and shape regulation.
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