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Method for Isolating Intact Nuclei

Technology #10-001-nagata

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Intact Nuclei isolated via the novel method developed by Nagata
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Researchers
Takako Nagata, Ph.D.
The George Washington University
Managed By
Brian Coblitz
Sr. Licensing Associate coblitz@gwu.edu (202) 994-4345
Patent Protection

Method for isolating nuclei

US Patent 8,546,134
Publications
Isolation of intact nuclei of high purity from mouse liver
Analytical Biochemistry, Volume 398, Issue 2, 15 March 2010, Pages 178–184

Current methods of nuclear isolation from liver disrupts the plasma membrane via homogenization and separation of the nuclei by ultracentifucation through gradients of sucrose or other substances for up to 80 min. The use of ultracentifugation for such a long time increases the potential for nuclear damage and degradation by endogenous proteases. The subject invention uses a conventional table top centrifuge instead of an ultracentrifuge. The centrifugation time, speed, force, and amounts of starting materials are far less than any of the conventional methods. The present invention also uses mild or high concentration buffers which are devoid of protease inhibitors. The buffers suspend the isolated sample after each step that gives the isolated sample a cushion and gives the best condition for nuclei separation and protects integrity and structures including nuclear inner membranes. Consequently, the nuclei separated from the cells are pure and intact without any damage and are ready to be used for a variety of biotechnological purposes.

The purity of the isolated nuclei was assessed biologically and morphologically, including analyses of representative marker proteins for nuclei and cytoplasm. The addition of collagenase to the procedure can be a viable option if priority is given to yield over nuclear purity and integrity. The subject method is especially suitable for small samples and so should facilitate studies with human liver biopsies and livers from experimental animals, especially mice, the most widely used species for gene targeting.

Applications

  • Isolation of nuclei having highest integrity and purity
  • Separation of intact nuclei from human and animal biopsies for diagnostic applications
  • Especially useful in gene targeting studies and other applications requiring intact nuclei

Advantages

  • Yields sufficient amounts of nuclei from only 0.1 to 10 g of cell samples.
  • Yields purified nuclei with intact inner nuclear membrane
  • Nuclei isolated are devoid of any cytoplasmic adulteration or cytoplasmic organelles
  • Nuclei retrieved are devoid of nuclear membranes or damaged endogenous nuclear material
  • Faster, simpler, easier, consistent results
  • Affordable method using conventional and easily available apparatus