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Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.
The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.
The goal of this protocol was to provide detailed steps to obtain mitochondria-enriched preparation from previously frozen heart tissue with a new technology of low energy mechanical disruption of tissue that improves tissue lysis and extraction of mitochondria. With this method, improved extraction efficiency without generating high shear stress or high temperature and short homogenization time (10-12 s) become achievable1.
To obtain mitochondria from archived frozen tissue is a valuable asset to perform both functional2 and biochemical studies3 otherwise not easily re....
Frozen offspring heart tissues were received from Dr. Sean Newcomer along with the institutional IACUC approval letter. The heart tissues were obtained from a long-term longitudinal study, flash-frozen in liquid nitrogen, and stored at -80 °C for future use. All protocols concerning the processing of offspring heart tissue followed the guidelines of Kansas City University IBC and IACUC committees.
1. Preparation of buffers and reagents
NOTE: Prepare all samp.......
Following the protocol, a good yield of mitochondria-enriched protein mixture from heart tissue was prepared. Approximately 15 mg/mL of mitochondria-enriched protein mixture was obtained from an average of 1.2 g frozen heart tissue harvested from the offspring of the sow. Observations indicated that less than 0.5 g of frozen heart tissue did not yield a sufficient amount of mitochondrial-enriched protein mixture to carry out a BN-PAGE assay. The amount of mitochondrial preparation was sufficient to perform (i) a standard.......
The critical steps for this protocol are indicated here. First, tissue homogenization should be carefully handled so that excessive sheer effects will not be applied during the tissue homogenization process. A tissue shredder should be used, which is a part of pressure cycling technology (PCT) for initial tissue homogenization9. This step will reduce the excessive stroke cycle of glass-on-glass homogenizer (Figure 1B) that may further destroy already fragile mitochond.......
This work was financially supported by Kansas City University's intramural grant for Abdulbaki Agbas and Summer Research Fellowship for Daniel Barrera. The authors are thankful for Dr. Jan Talley's editorial work.
....Name | Company | Catalog Number | Comments |
Amino caproic acid | Sigma/Aldrich | A2504-100G | |
Anti-Hu Total OxPhos complex kit | Invitrogen | 458199 | |
anti-VDAC antibody | abcam | ab15895 | use 1 µg/mL |
Coomassie G-250 | ThermoSientific | 20279 | |
Coomassie GelCode Blue | ThermoScientific | 24592 | |
Digitonin | Cabiochem | 300410 | |
Glass-Glass pestle homogenizer | VWR | KT885451-0020 | |
Image Studio | LICOR | ||
IR-Dye conjugated anti-Rabbit Ab | LICOR | LC0725 | |
Multiwell plate reader | BioTek | Synergy HT | |
Native molecular weight marker | ThermoFisher | BN2001 | |
Nylon mesh monofilament | Small Part Inc | CMN-74 | |
Orbital shaker | ThermoScientfic | ||
PCT Shredder | Pressure Bioscience Inc | ||
SEA BLOCK Blocking buffer | ThermoScienctific | 37527 | |
Shredder PULSE Tube | Pressure Bioscience Inc | FT500-PS | |
Table top centrifuge | Eppendorf | 5418 | |
Trypsin | Amresco | M150-1G | |
Trypsin inhibitor | Amresco | M191-1G | Requires fresh preparation |
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