Vesicle nuclear envelope preparation

The cytosol is divided into 0.1-ml samples, flash frozen, and stored at -80°C. A good cytosol preparation will not form nuclear envelopes when mixed with the sucrose-purified NEP-A fraction, nor will it contain vesicles that bind to the sperm chromatin. 

Numerous processes occurring during pronuclear formation in vitro, such as vesicle binding, vesicle fusion, and lamina assembly, rely on the function of specific membrane-associated proteins (Wilson and Newport, 1988 Collas and Poccia, 1996a,b for reviews, see Wiese and Wilson, 1993 Gerace and Foisner, 1994). This section describes procedures to prepare nuclear envelopes from in vitro sea urchin male pronuclei and to identify proteins in MVs suspected of playing a role in nuclear envelope assembly, as well as procedures for extracting peripheral membrane proteins. 

Zhu Y, Wang F, Zhang C, Du J. Preparation and mechanism insight of nuclear envelope-Uke polymer vesicles for facile loading of biomacromolecules and enhanced biocatalytic activity. ACS Nano 2014 8(7) 6644-54. 

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