Home › Cell Biology › Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting
Cell Biology JoVE (Open Access) Citable · DOI

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

DOI: 10.3791/59269-v
What you'll learn
  • ✓ Prepare mitochondrial lysates from cultured human cells
  • ✓ Execute blue native polyacrylamide gel electrophoresis (BN-PAGE)
  • ✓ Analyze respiratory chain complex assembly via immunoblotting
  • ✓ Interpret BN-PAGE results for OXPHOS complex characterization
Protocol

This protocol demonstrates the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis. Here the method applied to cultured human cells is described.

Difficulty
advanced
Total time
~4-6 hours per sample batch (includes lysis, gradient preparation, electrophoresis, and immunoblotting)
Model organism
Human cultured cells
Biosafety
BSL-1

Steps

1
Prepare mitochondrial lysates from cultured cells

Isolate and lyse mitochondria from cultured human cells to obtain respiratory chain complexes. This step involves cell harvesting, lysis buffer treatment, and centrifugation to obtain mitochondrial membrane protein extracts.

▶ 00:25
2
Assemble gradient gel for BN-PAGE analysis

Prepare polyacrylamide gradient gels suitable for blue native electrophoresis, including proper buffer systems and acrylamide concentrations to resolve respiratory complexes across molecular weight ranges.

▶ 02:40
3
Execute blue native gel electrophoresis protocol

Load prepared mitochondrial lysates onto BN-PAGE gels and perform electrophoresis under conditions that maintain native protein-protein interactions and complex integrity.

▶ 05:32
4
Analyze OXPHOS complex assembly patterns

Perform immunoblotting on BN-PAGE gels using respiratory chain complex-specific antibodies to visualize and characterize individual mitochondrial oxidative phosphorylation complexes.

▶ 06:40
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