Cells were harvested and washed once in isolation buffer (225 mM mannitol, 75 mM sucrose, 20 mM MOPS pH 7.2, 1 mM EGTA, 0.1% BSA). Cell pellets were then re-suspended and incubated in lysis buffer (100 mM sucrose, 10 mM MOPS pH 7.2, 1 mM EGTA, 0.1% BSA) for 5 min at 4 °C, and then homogenized with Omni homogenizer for 1 min at low speed. One volume of 1.25 M sucrose was then added, and the mixture was centrifuged at 1000 g for 10 min at 4 °C. The supernatant was transferred to a new tube, which was subjected to centrifuge at 12000 g for 5 min. The mitochondria-containing pellet was further purified by the Mitochondria Isolation kit (Miltenyi Biotec) according to manufacturer’s protocol. The eluted mitochondria were washed with high salt buffer (10 mM Tris-HCl pH 7.6, 10 mM KCl, 10 mM MgCl2, 0.4 M NaCl and 2 mM EDTA) and then resuspended in plasmid-safe reaction buffer (33 mM Tris-acetate pH 7.5, 66 mM potassium acetate, 10 mM magnesium acetate and 1 mM DTT). mtDNA was extracted from mitochondria by vigorous vortex followed by 2-3 stokes with 27 ½ G needle. Plasmid-safe ATP-dependent DNase (Epicentre) was added according to manufacturer’s protocol to digested contaminated nuclear DNA. The reaction was cleaned up by the DNA Clean & Concentrator kit (Zymo) to yield “pure” mitochondrial DNA.
Mitochondria isolation kit
Manufactured by Miltenyi Biotec
Sourced in Germany
The Mitochondria Isolation Kit is a laboratory equipment designed to efficiently isolate mitochondria from various cell types and tissues. This kit provides a straightforward protocol for the separation and purification of intact mitochondria, enabling researchers to study their structure, function, and biochemical properties.
Automatically generated - may contain errors
Lab products found in correlation
Ab92624, by Abcam (2 mentions)
Ab110413, by Abcam (2 mentions)
Total oxphos rodent wb antibody cocktail, by Abcam (2 mentions)
Aconitase activity assay kit, by Merck Group (2 mentions)
Ls6000sc, by Beckman Coulter (2 mentions)
Cell counter, by Beckman Coulter (2 mentions)
Hexokinase assay kit, by Abcam (2 mentions)
Anti tom70 antibodies, by Miltenyi Biotec (2 mentions)
Atp dependent dnase, by Illumina (1 mentions)
Dna clean concentrator kit, by Zymo Research (1 mentions)
Lsrfortessa x 20 flow cytometer, by BD (1 mentions)
Digitonin, by Merck Group (1 mentions)
Anti flag tag, by Fujifilm (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti gapdh 6c5, by Santa Cruz Biotechnology (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Hrp conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Ndufa9, by Abcam (1 mentions)
Ndufa9, by Merck Group (1 mentions)
Anti β actin ac 15, by Merck Group (1 mentions)
25 protocols using mitochondria isolation kit
1
Mitochondrial DNA Isolation Protocol
2
Assessing Mitochondrial Integrity via TMRE
3
Mitochondrial Protein Analysis in HEK293T Cells
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Mitochondrial fractions were isolated from WT and OSGEPL1 KO HEK293T cells (1 × 107 cells) using the Mitochondria Isolation Kit (Miltenyi Biotec. K.K.) and subjected to western blotting to measure steady-state levels of protein components in respiratory chain complexes using the Total OXPHOS Rodent WB Antibody Cocktail (an antibody mixture targeting ATP5A, UQCRC2, MTCO1, SDHB, and NDUF88; ab110413, Abcam), an anti-ND5 antibody (ab92624, Abcam), and an anti-ND2 antibody (19704-1-AP, Proteintech). Other antibodies used in this study were as follows: anti-OSGEPL1 (25694-1-AP, Proteintech), anti-GAPDH (6C5, Santa Cruz Biotech), anti-FLAG-tag (1E6, Wako), HRP-conjugated donkey anti-mouse/rabbit IgG (715-035-150/715-035-152, Jackson ImmunoResearch), anti-FLAG M2 affinity gel (A2220, Sigma), and Alexa Fluor 488-conjugated goat anti-mouse IgG (A-11001, ThermoFisher). To monitor YRDC subcellular localization using the anti-FLAG-tag antibody, mitochondrial fractions were carefully washed with 2 mg/mL digitonin (Sigma) to remove the outer membrane with cytoplasmic components.
4
Mitochondrial Respiratory Chain Complex Analysis
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The mitochondrial fraction was isolated from WT and GTPBP3 KO cells (1 × 107 cells) using the Mitochondria Isolation Kit (Miltenyi Biotec). Subunit proteins of mitochondrial respiratory chain complexes were detected by immunoblotting using Total OXPHOS Rodent WB Antibody Cocktail (1:250, ab110413, Abcam), anti-ND2 antibody (1:200, 19704–1-AP, Proteintech) and anti-mt-VDAC1 antibody (1:500, 10886–1-AP, Proteintech) as a control. HRP-conjugated anti-rabbit IgG (1:20000, 715–035-152, Jackson ImmunoResearch) was used as the secondary antibody.
5
Mitochondrial Isolation from B Cells
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Mitochondria were isolated from B cells using Mitochondria isolation kit (Miltenyi Biotec)
following manufacturer's instructions with some modifications. Briefly, frozen B cells
(2–10 × 107 cells) were directly resuspended in pre-cooled phosphate-buffered
saline (1 ml per 108 total cells) supplemented with ethylenediamine tetraacetatic
acid (EDTA; 2 mM), anti-protease and –phosphatase inhibitors and benzonase
(50 U), and incubated for 20 min at 4 °C. Cell homogenization was performed
with a 26-G needle stepwise using 5–10 stokes. Lysates were diluted to 1 × separation
buffer (Miltenyi Biotec) and proceeded to magnetic labeling by incubation with anti-Tom22 magnetic
beads for 1 h at 4 °C on a wheel. The labeled cell lysate was loaded on a MACS
column placed in a magnetic field and let run through. Lysates were re-loaded three times. Column
was then intensively washed out and the magnetically labeled mitochondria were then flushed out.
Pre- and post-mitochondria-purified fractions were separated by SDS-PAGE and the presence of
cytoplasmic β-actin protein, Mt hsp60 or NDUFA9 proteins, and nuclear KDM1a were
evaluated by western blot analysis using monoclonal anti-β-actin (AC-15,
Sigma-Aldrich), -hsp60 (Biosciences Inc., Allentown, PA, USA), -NDUFA9 (Abcam, Cambridge, UK) and
KDM1a (Cell Signaling Technology).
following manufacturer's instructions with some modifications. Briefly, frozen B cells
(2–10 × 107 cells) were directly resuspended in pre-cooled phosphate-buffered
saline (1 ml per 108 total cells) supplemented with ethylenediamine tetraacetatic
acid (EDTA; 2 mM), anti-protease and –phosphatase inhibitors and benzonase
(50 U), and incubated for 20 min at 4 °C. Cell homogenization was performed
with a 26-G needle stepwise using 5–10 stokes. Lysates were diluted to 1 × separation
buffer (Miltenyi Biotec) and proceeded to magnetic labeling by incubation with anti-Tom22 magnetic
beads for 1 h at 4 °C on a wheel. The labeled cell lysate was loaded on a MACS
column placed in a magnetic field and let run through. Lysates were re-loaded three times. Column
was then intensively washed out and the magnetically labeled mitochondria were then flushed out.
Pre- and post-mitochondria-purified fractions were separated by SDS-PAGE and the presence of
cytoplasmic β-actin protein, Mt hsp60 or NDUFA9 proteins, and nuclear KDM1a were
evaluated by western blot analysis using monoclonal anti-β-actin (AC-15,
Sigma-Aldrich), -hsp60 (Biosciences Inc., Allentown, PA, USA), -NDUFA9 (Abcam, Cambridge, UK) and
KDM1a (Cell Signaling Technology).
6
Purification and Extraction of RNA Subtypes
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The mRNA was purified by Ambion Dynabeads® mRNA DIRECT™ Purification Kit twice according to according to the manufacturer's instruction. For rRNA, total RNAs were separated with 1.5% agarose gel, then 18S and 28 S rRNA were recovered from the gel by Zymoclean™ Gel RNA Recovery Kit (Zymo Research, Orange, CA, USA). For mtRNA, the mitochondria were separated by use of the magnetic beads method (Mitochondria Isolation Kit; Miltenyi Biotec, Auburn, CA, USA). Total mtRNA was extracted by Trizol.
7
Mitochondrial Aconitase Activity Assay
8
Mitochondrial Protein Synthesis Assay
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Mitochondria were isolated from BM Lin− cells using the Mitochondria Isolation kit (Miltenyi Biotech Inc) following manufacturer instructions. Cell-free mitochondrial translation was carried out using a modified protocol described previously (McKee et al., 2006 (link)). Briefly, mitochondria were incubated at 3 mg protein/ml in 30 μL protein synthesizing medium. The mitochondrial mix was incubated at 30 °C in the presence of vehicle (DMSO) or the mitochondrial translation inhibitor Chloramphenicol (CAP; 10 μM) for 5 min, and 4 μL (4 μCi) [3H]-leucine was then added to the mix and incubation continued for 60 min. The incorporation of [3H]-leucine into mitochondrial protein was determined by counting the [3H]-leucine-labeled proteins in a Beckman LS6000SC liquid scintillaton counter.
9
Mitochondrial Citrate Tracing Assay
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Mitochondria were prepared with the magnetic beads method (Mitochondria Isolation Kit; Miltenyi Biotec,), and the resulting mitochondrial pellets were reconstituted in assay buffer (125 mM KCl, 10 mM Tris/MOPS, 0.1 mM EGTA/Tris, 1 mM Pi, pH 7.4) supplied with indicated nutrients and tracer3 (link). For citrate tracing, 40 µM [U-13C]-citrate, 40 µM NADH, and 40 µM CoA with or without 40 µM ATP and 50 µM ACLY inhibitor were added to the assay buffer. Mitochondria were incubated in the tracing buffer for 10 min, at 37 °C with 200 r.p.m. agitation in a heat block.
10
Mitochondrial Aconitase Activity Assay
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For mitochondrial activity tests, mitochondria were isolated from 104–105 HEK293T cells using the mitochondria isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions. Mitochondrial activity was then assessed by determining the mitochondrial aconitase activity (Aconitase Activity Assay Kit, Merck) according to the manufacturer’s instructions. Values were normalized to the mitochondrial protein concentration.
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