Aug 21, 2025
Electron Transport Chain Protocol
- Ashley Seifert1,
- Hemendra Vekaria2,3,
- Ebenezer Aryee4
- 1University of Kentucky, Department of Biology;
- 2Medical University of South Carolina;
- 3Department of Neurosurgery;
- 4University of Kentucky
Protocol Citation: Ashley Seifert, Hemendra Vekaria, Ebenezer Aryee 2025. Electron Transport Chain Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrw39olmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 17, 2025
Last Modified: August 21, 2025
Protocol Integer ID: 224834
Keywords: ASAPCRN, electron transport chain, mitochondria, mitochondrial dysfunction in frozen brain sample, mitochondrial respiration in frozen biological sample, mitochondrial dysfunction, mitochondrial respiration, analyzing electron transport chain activity, electron transport chain activity of complex, electron transport chain protocol, electron transport chain protocol this protocol, frozen brain sample, respirometry in frozen sample, frozen biological sample, respirometry, traumatic brain injury, seifert lab
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-020495
NIH
Grant ID: R01AR070313
Abstract
This protocol describes the procedure for analyzing Electron Transport Chain activity of Complexes 1, 2, and 4 in mitochondria isolated from fibroblasts used by the Seifert Lab. It follows the "Electron Transport Chain Protocol" (ETCP) described in:
Vekaria, H.J., Kalimon, O.J., Prajapati, P., Velmurugan, G.V., Sullivan, P.G., 2024. An efficient and high-throughput method for the evaluation of mitochondrial dysfunction in frozen brain samples after traumatic brain injury. Front Mol Biosci 11, 1378536.
which is a modified version of the "Respirometry in Frozen Samples" (RIFS) assay from:
Acin-Perez, R., Benador, I.Y., Petcherski, A., Veliova, M., Benavides, G.A., Lagarrigue, S., Caudal, A., Vergnes, L., Murphy, A.N., Karamanlidis, G., Tian, R., Reue, K., Wanagat, J., Sacks, H., Amati, F., Darley-Usmar, V.M., Liesa, M., Divakaruni, A.S., Stiles, L., Shirihai, O.S., 2020. A novel approach to measure mitochondrial respiration in frozen biological samples. Embo j 39, e104073.
Troubleshooting
Thaw frozen mitochondrial samples
Add 25 µL Mitochondria Isolation Buffer (MIB; 215 millimolar (mM) mannitol, 75 millimolar (mM) sucrose, 0.1% BSA, 20 millimolar (mM) HEPES, and 1 millimolar (mM) EGTA, adjusted to pH 7.2 with KOH)
Gently resuspend pellet
Measure concentration of 4 µL of each mitochondrial mix by BCA
Prepare
Equipment
Seahorse XFe96 Analyzer
NAME
Agilent
BRAND
BLE2100298
SKU
LINK
96-well cartridge ports as follows:
Port A (ETC 1): 3220 µL Mitochondrial Respiration buffer w/o BSA, 2.8 µL Rotenone, 280 µL succinate;
Port B: 3500 µL Mitochondrial Respiration buffer w/o BSA, 3.5 µL Antimycin A;
Port C: 3325 µL Mitochondrial Respiration buffer w/o BSA, 88 µL Ascorbate, 88 µL TMPD;
Port D: 3033 µL Mitochondrial Respiration buffer w/o BSA, 480 µL Azide.
Load cartridge well ports with 25 µL of port mixes
On ice Load 75 µL of mitochondrial sample (2 µg protein) per well
Balance the plate and spin at 3214 rcf, 4°C, 00:10:00
Prepare 11 mL Alamethicin Assay solution (10.6 mL Mitochondrial Respiration buffer w/o
BSA, 40 mg/mL Alamethicin 9.63 µL , 1.5 millimolar (mM) NADH 385 µL , 10 micromolar (µM) cytochrome c 48 µL )
Add 100 µL assay solution to each well
Run plate on
Equipment
Seahorse XFe96 Analyzer
NAME
Agilent
BRAND
BLE2100298
SKU
LINK
sequentially injecting the contents of Ports A-D over 01:00:00
Protocol references
Vekaria, H.J., Kalimon, O.J., Prajapati, P., Velmurugan, G.V., Sullivan, P.G., 2024. An efficient and high-throughput method for the evaluation of mitochondrial dysfunction in frozen brain samples after traumatic brain injury. Front Mol Biosci 11, 1378536.