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Preparation of nuclear matrices from cultured cells: subfractionation of nuclei in situ

Staufenbiel, Matthias and Deppert, Wolfgang (1984) Preparation of nuclear matrices from cultured cells: subfractionation of nuclei in situ. Journal of Cell Biology, 98 (5). pp. 1886-1894. ISSN 0021-9525. http://resolver.caltech.edu/CaltechAUTHORS:20120629-142622097

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Abstract

Analyses of the different structural systems of the nucleus and the proteins associated with them pose many problems. Because these systems are largely overlapping, in situ localization studies that preserve the in vivo location of proteins and cellular structures often are not satisfactory. In contrast, biochemical cell fractionation may provide artifactual results due to cross-contamination of extracts and structures. To overcome these problems, we have developed a method that combines biochemical cell fractionation and in situ localization and leads to the preparation of a residual cellular skeleton (nuclear matrix and cytoskeletal elements) from cultured cells. This method's main feature is that cell fractionation is performed in situ. Therefore, structures not solubilized in a particular extraction step remain attached to the substrate and retain their morphology. Before and after each extraction step they can be analyzed for the presence and location of the protein under study by using immunological or cytochemical techniques. Thereby the in vivo origin of a protein solubilized in a particular extraction step is determined. The solubilized protein then may be further characterized biochemically. In addition, to allow analyses of proteins associated with the residual cellular skeleton, we have developed conditions for its solubilization that do not interfere with enzymatic and immunological studies.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1083/jcb.98.5.1886 DOIUNSPECIFIED
http://jcb.rupress.org/content/98/5/1886.abstractPublisherUNSPECIFIED
Additional Information:© 1984 Rockefeller University Press. Received for publication 7 November 1983; Published May 1, 1984. We thank Ms. Petra Epple for expert help during this study. In addition, we gratefully acknowledge Dr. R. Martin and the members of the Section of Electron Microscopy at the University of Ulm for their help with the electron microscopy. This study was supported by grants from the Stiftung Volkswagenwerk (VW I/37084) and from the Deutsche Forschungsgemeinschaft (De 212/5-3) . Empigen BB was a generous gift of the Marchon Division of Albright and Wilson, Ltd.
Funders:
Funding AgencyGrant Number
Stiftung VolkswagenwerkVW I/37084
Deutsche Forschungsgemeinschaft (DFG)De 212/5-3
Marchon Division of Albright and Wilson Ltd.UNSPECIFIED
Record Number:CaltechAUTHORS:20120629-142622097
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20120629-142622097
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:32215
Collection:CaltechAUTHORS
Deposited By: Jason Perez
Deposited On:02 Jul 2012 14:43
Last Modified:26 Dec 2012 15:27

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