Home › Microbiology › Rapid Isolation of the Mitoribosome from HEK Cells
Microbiology JoVE (Open Access) Citable · DOI

Rapid Isolation of the Mitoribosome from HEK Cells

DOI: 10.3791/57877-v
What you'll learn
  • ✓ Isolate intact mitochondria from HEK suspension cells using differential centrifugation
  • ✓ Purify mitoribosomes from isolated mitochondria via density gradient separation
  • ✓ Verify mitoribosome integrity and purity using analytical techniques
  • ✓ Apply native compartment extraction methods to study organellar ribosomes
Protocol

Biopharma Insights Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

Difficulty
advanced
Total time
~3–4 hours per sample batch
Model organism
HEK293
Biosafety
BSL-1

Steps

1
Isolate mitochondria from HEK suspension cells

Harvest HEK cells and perform differential centrifugation steps to obtain a purified mitochondrial pellet. Use cell lysis and successive spins to separate mitochondria from cytoplasmic and nuclear debris.

▶ 00:53
2
Purify mitoribosomes from mitochondrial lysate

Lyse isolated mitochondria and separate mitoribosomes from other mitochondrial components using density gradient centrifugation. Collect the mitoribosome-containing fraction based on sedimentation profile.

▶ 05:15
3
Assess isolated mitoribosome quality and yield

Characterize the purified mitoribosomes using appropriate analytical methods to confirm structural integrity, purity, and functional properties. Document morphology and composition of the isolated particles.

▶ 08:12
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