PURE and EZ 'Nucleisolation'

Purifed nuclei Transcription run-off experiments and nuclear component studies require nuclei that are functionally intact. The isolation of such nuclei has traditionally been achieved through detergent-based cell lysis followed by fractionation via differential centrifugation. Sigma-Aldrich of St. Louis now offers two products--the Nuclei EZ Prep and Nuclei PURE Prep nuclei isolation kits--for the isolation of functionally active nuclear components. According to senior R&D scientist Ned Watson,

Jan 8, 2001
Aileen Constans


Purifed nuclei
Transcription run-off experiments and nuclear component studies require nuclei that are functionally intact. The isolation of such nuclei has traditionally been achieved through detergent-based cell lysis followed by fractionation via differential centrifugation. Sigma-Aldrich of St. Louis now offers two products--the Nuclei EZ Prep and Nuclei PURE Prep nuclei isolation kits--for the isolation of functionally active nuclear components. According to senior R&D scientist Ned Watson, who developed the kits, the EZ Prep and PURE Prep methods are application-tested and offer investigators an easy-to-follow protocol for nuclei isolation.

The EZ Prep kit was developed for the isolation of nuclei from tissue culture cell lines. Unlike the standard protocol, which involves the harvesting of adherent cells by scraping in a phosphate-buffered saline followed by lysis, the EZ Prep method lyses and harvests cells at the same time. This modification produces higher yields than the traditional method and also eliminates the problems associated with harvesting by scraping--namely, the inclusion of damaged cells and cell debris in the lysate. In addition, the process removes the outer membrane of the nucleus while leaving the nucleus itself intact, thus eliminating contamination by cytoplasmic components such as the rough endoplasmic reticulum (because the procedure removes the outer membrane, nuclei isolated with these kits cannot be used for nuclear transport studies). Nuclei are then separated from other cell components by differential centrifugation. With the Nuclei PURE Prep kit, nuclei can be isolated from animal tissues or from difficult-to-lyse cultured cells. This method is similar to that of the EZ Prep kit, but it involves a step in which the cells are spun by ultracentrifugation through a dense (1.8 M) sucrose cushion. According to Watson, a typical EZ Prep isolation can be achieved in an hour; the process is slightly longer for the PURE Prep procedure due to the additional sucrose cushion purification step.

In addition to these kits, Sigma-Aldrich offers the Nu-CLEAR Extraction Kit for the preparation of nuclear extracts that can be used for the analysis of transcription factors by electrophoretic mobility shift assay and for in vitro transcription reactions.

--Aileen Constans (aconstans@the-scientist.com)

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