Figure 1.
Efficient mitochondrial isolation from mouse tissues using the anti-TOM22 magnetic beads method.
Proteins of wash (W) and elution (E) fractions were compared by Western blot analysis from A heart, B brain and C liver tissues. Anti-TOM20 and anti-cytochrome c antibodies were used to detect mitochondrial marker proteins (mit). Anti-PEX1 and PDI antibodies were used to detect peroxisomal (per) or endoplasmic reticulum (ER) marker proteins, respectively.
Figure 2.
The anti-TOM22 magnetic beads method yields intact mitochondria.
Anti-TOM22 magnetic beads method was applied to purify mitochondria from A heart, B skeletal muscle, C brain and D liver tissues. Mitochondria were isolated by differential centrifugation technique from E liver tissue. Mitochondrial morphology was investigated by transmission electron microscopy from four biological samples per group and representative pictures are shown. Pictures were taken at 4,000× (left panel) and 20,000× (right panel) magnification and black bars display 1 µm (left panel) and 200 nm (right panel), respectively. Black dots at the outer membrane in A, B, C and D pictures represent anti-TOM22 magnetic beads.
Figure 3.
Liver, muscle and brain mitochondria isolated using anti-TOM22 magnetic beads show reliable mitochondrial function.
Oxygen consumption of isolated A liver, B muscle and C brain mitochondria were measured by a Clark electrode. Malate+pyruvate (MP) and malate+glutamate (MG) served as specific substrates for complex I, succinate (S, for complex II and glycerol-3-phosphate (GP) for complex III, respectively. ADP was given to malate+pyruvate to determine state III respiration via complex I. Oligomycin and CCCP were used with succinate to assess state IVo and uncoupled respiration, respectively. The given oxygen consumption units are nmol oxygen/min/mg mitochondria. Data are represented as means of three independent experiments ± SD.
Figure 4.
Liver mitochondria isolated by magnetic beads show less non-mitochondrial proteins than mitochondria isolated by differential centrifugation.
A Mitochondrial preparation obtained by differential centrifugation (DC) was compared to anti-TOM22 magnetic beads (MB) isolation by Western blot using antibodies against mitochondrial (TOM20 and cytochrome c) and ER (PDI) marker proteins. B–D Proteins were identified by mass spectrometry and averages of eight samples per group were calculated for MB and DC mitochondrial fractions, respectively. The relative abundance is represented as a linear ratio (average of MB/average of DC). When these ratios were less than 1 (DC average>MB average) the negative inverse ratio was taken and the values were transposed by the equation: −1/(MB/DC ratio). Positive ratios indicate the presence of higher protein levels in the MB fraction, whereas negative ratios indicate higher protein levels in the DC fraction. Diagram titles indicate the applied GO terms, which were identified by Genomatix software GO term analysis. Statistical analyzes were made by Mann-Whitney U test and p values were corrected to multiple testing according to Benjamini&Hochberg [23]. A false discovery rate (FDR) <10% was set as the significance level and significant proteins are denoted by stars. Numbers are given for every tenth protein and the names of the identified proteins are summarized in Table S1. ETC: electron transport chain.
Figure 5.
Liver mitochondria isolated by magnetic beads have higher oxygen consumption rates than mitochondria isolated by differential centrifugation.
Oxygen consumption rates (OCRs) were determined using an XF24 Extracellular Flux Analyzer with the given amount of freshly isolated mitochondria (µg/well), which were purified by A DC or B MB methods. Briefly, succinate was used as a substrate to feed electrons to complex II and basal (state II) respiration was measured. State III respiration was determined in the presence of ADP. Oligomycin (Oligo) addition blocked ATP synthase and so evoked state IVo respiration. Maximum respiration (state uncoupled) was measured in the presence of the uncoupler FCCP. Finally, Antimycin A (Antim) was given to inhibit complex III. Data are represented as means of three independent experiments (5–8 replicates per groups) ± SD.