(M1130-06-35) Maintaining Pristine Structure for High-Purity Isolates: A Combined Method for Nuclei and Mitochondria

Purpose: Current methods for isolating nuclei and mitochondria from cells or tissues rely on homogenization, enzymatic digestion, and high-speed centrifugation; potentially damaging those organelles and degrading proteins. These methods also require extended processing times. We aimed to develop a simpler and gentler method for isolating intact-and-pure nuclei and mitochondria simultaneously from one sample.

Methods: This methodology doesn’t need either protein inhibitors or trypsin. All the procedures are done on ice within one hour. Tissues are mashed through a cell strainer with a 40-micrometer pore size. Cell suspensions are processed only by microcentrifuge. Nuclei and mitochondria are isolated from single sample in parallel, and their purity and integrity were evaluated using specific protein markers (Figure 1) and morphological analysis (Figure 2 and 3).

Results: These isolated nuclei and mitochondria are shown intact with high purity by electron micrography and biochemical analysis. Also, this protocol significantly reduced processing time compared to conventional protocols.

Conclusion: This optimized method offers a faster and gentler approach for isolating intact organelles with high purity from cells and tissues. This is particularly valuable for analyzing smaller samples, such as human biopsies and materials from small animals like mice, for genetic and kinetic studies.

References: Nagata T, Redman RS, Lakshman R. Isolation of intact nuclei of high purity from mouse liver. Anal Biochem. 2010;398(2).

Acknowledgements: Competing interests - The author declares no competing interests.

Funding: This work was supported by NT Biologics.


Figure 1. Western blotting with lamin B1 and cytochrome c and actin.


Figure 2. Transmission electron micrographs of the isolated nuclei.


Figure 3. Transmission electron micrographs of isolated mitochondria.