Preparation of HeLa cell extract

This procedure describes the preparation of nuclear and cytoplasmic extracts from cells grown in suspension (2-201) and is essentially as described by Dignam et al.1 and modified by Jamison and Garcia-Bianco.2 A procedure for small-scale preparation of extracts from HeLa cells grown as monolayer has been described by Lee and Green.3 The latter procedure is recommended when the cell material is scarce, if radioactively labelled extracts are made, if several extracts from parallel cultures, or if expensive growth conditions are necessary for cell propagation. 

Both methods involve swelling of the cells in hypotonic solution, disruption of the plasma membrane, and extraction of the nuclei in a high salt buffer. The extracts support a number of nuclear functions and have been extremely useful in studying transcription and splicing (see Section 5.1). Nuclear extracts are also a useful starting point for purification of snRNAs, snRNPs, hnRNP proteins and SR-proteins (Section 3.1.2). 

nuclei, or nuclear extracts are also commercially available from various sources. Material from The National Cell Culture Center, 8500 Evergreen Boulevard, Minneapolis, MN 55433 USA and Computer Cell Culture Center sa, Place du Parc, 20 B-7000 Mons, Belgium, is recommended. 

50-ml disposable screw-cap tubes and swinging bucket rotor 15-ml tubes, 50-ml polycarbonate tubes and Beckman JA20 rotor (or equivalent) 

SW28 rotor and polyallomer tubes (or equivalent) if preparing S100 extract 

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In addition, we present methods for the preparation of HeLa cell extracts that can support import in vitro. In general, we find for both proteins and snRNPs that the Xenopus egg extract is the most efficient in terms of the speed of nuclear import and the final level of intranuclear accumulation. The Xenopus oocyte extract, HeLa cell extract, and reticulocyte lysates are generally less efficient in our hands. We have not been able to reconstitute nuclear import using extracts from Drosophila early embryos (C. Dingwall, unpublished). 

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