Ms604

Manufactured by Abcam

Sourced in United States

The MS604 is a liquid chromatography-mass spectrometry (LC-MS) system. It is designed for the analysis of small molecules. The system includes a high-performance liquid chromatography (HPLC) unit and a mass spectrometer. This equipment is used for the separation, detection, and identification of chemical compounds in complex mixtures.

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Lab products found in correlation

11 protocols using ms604

Western Blotting and Immunohistochemistry Protocols

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For western blotting, primary antibodies against Drp1 (1:1000, ab56788), Mfn1 (1:500, ab57602), Mfn2 (1:1000, ab56889), GAPDH (1:3000, ab8245), p62 (1:1000, ab56416), LC3 I/II (1:1000, ab128025), Fgf21 (1:1000, ab171941), LONP1 (1:500, ab103809), Hsp60 (1:2000, ab46798), and OXPHOS (a premixed cocktail of antibodies against CI-NDUFB8, CII-SDHB, CIII-UQCRC2, CIV-MTCO1 and CV-ATP5A, 1:500, MS604) were from Abcam. Primary antibody against AFG3L2 (1:500, sc-84687) was from Santa Cruz Biotechnology. Horseradish peroxidase (HRP) linked secondary antibodies anti-mouse IgG (1:3000, cs7076) and anti-rabbit IgG (1:3000, cs7074) were from Cell Signaling Technology.
For immunohistochemistry, primary antibodies against C5b-9 (1:200, ab55811) and heavy chain cardiac Myosin (1:200, ab15) were from Abcam. Alexa Fluor conjugated secondary antibodies anti-mouse IgG (1:400, A-11029) and anti-rabbit IgG (1:400, A-11035) were from Invitrogen.

Song M., Mihara K., Chen Y., Scorrano L, & Dorn GW I.I. (2015). Mitochondrial fission and fusion factors reciprocally orchestrate mitophagic culling in mouse hearts and cultured fibroblasts. Cell metabolism, 21(2), 273-285.

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Mitochondrial Metabolism Profiling in DKO Mice

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Using immunoblots we determined the levels of hexokinase 1 and 2 (HK 1 and 2), lactate dehydrogenase (LDH), pyruvate kinase M1 and M2 (PK M1and M2), Glyceraldehyde 3 phosphate dehydrogenase (GAPDH), mitochondrial electron transport chain [26 (link)] subunit proteins, mitofusin 2 (Mfn 2), dynamin related protein (Drp1), citrate synthetase (CS), mitochondrial transcription factor A (TFAM), AMP activated kinase (AMPK), phosphorylated AMPK (pAMPK) in the muscles of DKO and WT mice. Tissue homogenates were separated using standard SDS—PAGE gels; transferred to nitrocellulose membrane and immunoprobed with specific primary antibodies; HK 1, HK 2 PK M1, PK M2, AMPK, pAMPK (glycolysis antibody sampler kit -8337 (1:1000)), AMPKα, p-AMPKα (Cell signaling AMPK and ACC sampler kit -9957 (1:1000)); GAPDH (Fitzgerald 10R-G109A (1:5000)), LDH (Santa Cruz sc33781 (1:2000)), Mfn 2 (Santa Cruz sc50331 (1:500)), TFAM (Santa Cruz sc23588 (1:500)), (Mitochondrial ETC proteins- (Mitobiosciences MS604 (1:500)), CS, (Abcam ab129095 (1:1000)) Drp 1 (Abcam ab56788 (1:1000)) followed by horseradish peroxidase-conjugated secondary antibody (1:25,000–1:50,000). The antibody dilutions were as per manufacturer’s protocol. Signals were detected by WestDura substrate (Pierce) and quantified by densitometry (ImageJ 1.41o program). GAPDH was used as loading control for all the quantifications.

Pant M., Sopariwala D.H., Bal N.C., Lowe J., Delfín D.A., Rafael-Fortney J, & Periasamy M. (2015). Metabolic Dysfunction and Altered Mitochondrial Dynamics in the Utrophin-Dystrophin Deficient Mouse Model of Duchenne Muscular Dystrophy. PLoS ONE, 10(4), e0123875.

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Immunofluorescence Staining of Immune Cells

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After red blood cells lysis, blood cells were cytospun onto SuperFrost Plus slides. Samples were fixed in 10% formalin (Sigma–Aldrich) and then stained for CD14 (13-0149-82, Invitrogen) overnight and with the appropriate secondary antibody (Streptavidin AF 647, S32357, ThermoFisher Scientifc). Samples were then permeabilised and stained for IRF5 (10547-1-AP, Proteintech) and OXPHOS (MS604, Abcam) with the appropriate secondary antibodies (goat anti-mouse FITC (A11001) and anti-rabbit AF555 (A21428), Invitrogen). Nuclei were counterstained with Hoescht 33342 (ThermoFisher Scientific). Images were acquired with a confocal microscope (Zeiss LSM 710) and analysed with ImageJ (Fiji).
Adipose tissue sections were stained for Mac2 (CL8942AP, Cedarlanelabs) overnight and then with the appropriate secondary antibody. Nuclei were counterstained with Hoescht 33342 (ThermoFisher Scientific). Slides were scanned using Zeiss Axio Scan Z1, and Mac2 staining was quantified with Visiopharm.

Orliaguet L., Ejlalmanesh T., Humbert A., Ballaire R., Diedisheim M., Julla J.B., Chokr D., Cuenco J., Michieletto J., Charbit J., Lindén D., Boucher J., Potier C., Hamimi A., Lemoine S., Blugeon C., Legoix P., Lameiras S., Baudrin L.G., Baulande S., Soprani A., Castelli F.A., Fenaille F., Riveline J.P., Dalmas E., Rieusset J., Gautier J.F., Venteclef N, & Alzaid F. (2022). Early macrophage response to obesity encompasses Interferon Regulatory Factor 5 regulated mitochondrial architecture remodelling. Nature Communications, 13, 5089.

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Mitochondrial Protein Analyses by Western Blot

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All protein samples were lysed by homogenization in RIPA buffer. Samples
were run by electrophoresis on polyacrylamide Tris-glycine SDS gels. The
following antibodies were used in the study: NCLX (1:500, NCKX6 Santa Cruz,
sc-161921); MCU (1:1,000, Sigma-Aldrich, HPA016480); MCUb (1:1,000, Abgent,
AP12355b); MICU1 (1:500, custom generation by Yenzyme); EMRE (1:250, Santa Cruz,
sc-86337); LETM1 (1:1,000, Proteintech, 16024-1-AP); VDAC (1:2,500, Abcam,
ab15895); cyclophilin D (1:5,000, Abcam, ab110324); PDH subunits (1:1,000,
Abcam, ab92696), p-PDH^S293 (1:1,000, Abcam, ab110330) ETC respiratory
chain complexes (1:5,000, OxPhos Cocktail, Abcam, MS604) and Licor IR secondary
antibodies (1:12,000). All blots were imaged on a Licor Odyssey system
(anti-mouse, 926-32210; anti-rabbit, 926-68073; anti-goat, 926-32214). Western
blot details have been previously reported in detail^{13 (link)}. All source gels (that is, full-length
western blots) and band density results are available in Supplementary Fig. 1.

Luongo T.S., Lambert J.P., Gross P., Nwokedi M., Lombardi A.A., Shanmughapriya S., Carpenter A.C., Kolmetzky D., Gao E., van Berlo J.H., Tsai E.J., Molkentin J.D., Chen X., Madesh M., Houser S.R, & Elrod J.W. (2017). The mitochondrial Na+/Ca2+ exchanger is essential for Ca2+ homeostasis and viability. Nature, 545(7652), 93-97.

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Protein Expression Analysis in Cells

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Equal amounts of proteins were separated using SDS-PAGE under reducing conditions. Proteins of interest were detected using the following antibodies; SIRT1 (S5196, Sigma-Aldrich), SIRT3 (5490S, Cell signaling), SIRT6 (GTX105611, Gene Tex), GLUT4 (sc53566, Santa Cruz Biotechnology), HK2 (2857, Cell signaling), PKM1/2 (3190, Cell signaling), PDHA1 (3205, Cell signaling), ACADVL (ab155138, Abcam), ACADL (ab82853, Abcam), CD36 (ABM-5525, Cascade Bioscience), AMPKα (G. Hardie, University of Dundee, Dundee, Scotland, UK), pAMPKα T172 (2531, Cell signaling), ACC (3661, Cell signaling), pACC S79 (3662, Cell signaling), PGC-1α (3242, EMD Millipore), ATP5A, UQCRC2, MTCO1, SDHB, NDUFB8 (MS604, MitoSciences), COX4 (4850P, Cell signaling), CYTC (11940, Cell signaling), eEF2 (2332, Cell signaling), Akt2 (2964, Cell signaling), pAkt S473 (9271, Cell signaling), pAkt T308 (9275, Cell signaling), GSK3α/β (5676, Cell signaling), pGSK3α/β S21/S9 (9331, Cell signaling). Densitometric analysis of immunoblots was performed on four or seven individual samples using Image Lab Software (Bio-Rad Laboratories), and a representative selection is presented in each figure.

Svensson K., LaBarge S.A., Martins V.F, & Schenk S. (2017). Temporal overexpression of SIRT1 in skeletal muscle of adult mice does not improve insulin sensitivity or markers of mitochondrial biogenesis. Acta physiologica (Oxford, England), 221(3), 193-203.

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Mitochondrial Function Assessment in Fibroblasts

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Protein extract from fibroblasts was separated by electrophoresis on a 4–12% polyacrylamide gel (NuPAGE). Immunoblotting was performed as described using antibodies to SSBP1, (12212-1-AP, Proteintech), mitochondrially encoded cytochrome c oxidase 1 (MT-CO1, ab14705, Abcam), mitochondrially encoded cytochrome c oxidase 2 (MT-CO2, 12C4F12, Abcam), succinate dehydrogenase complex flavoprotein subunit A (SDHA, ab14715, Abcam), ATP synthase subunit alpha (ATP5A, MS604, Mitosciences), translocase of outer membrane 20 (TOMM20, ab56783, Abcam) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-25778, Santa-Cruz) (Pfeffer et al., 2014 (link)). Cellular oxygen consumption rate (OCR) was assayed using the Seahorse XF96 Extracellular Flux Analyser with sequential addition of oligomycin (1 μM), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 1 μM) and rotenone/antimycin (1 μM) to measure basal and maximal respiration. Cell growth was assessed by growing fibroblast cells with high glucose medium versus glucose-free medium supplemented with 5 mM galactose using IncuCyte® Live Cell imager, Essen Bioscience.

Kullar P.J., Gomez-Duran A., Gammage P.A., Garone C., Minczuk M., Golder Z., Wilson J., Montoya J., Häkli S., Kärppä M., Horvath R., Majamaa K, & Chinnery P.F. (2017). Heterozygous SSBP1 start loss mutation co-segregates with hearing loss and the m.1555A>G mtDNA variant in a large multigenerational family. Brain, 141(1), 55-62.

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Muscle Triglycerides and Protein Analysis

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Muscle samples were frozen immediately in liquid nitrogen once harvested from mice and were stored in −80 °C. For Western blot, tissues were cut on dry ice into small pieces and were lysed in RIPA buffer supplemented with PMSF, proteinase inhibitors, and phosphatase inhibitors. Primary antibodies include Phospho-Akt (Ser473) (CST 736E11, #3787), Akt (CST #9272), Hsp90 (CST #4874), Ran (CST #4462), and OXPHOS proteins (MitoSciences MS604). Secondary antibodies include mouse anti-rabbit IgG-HRP (sc-2357) and m-IgGx BP-HRP (sc-516102). For measuring muscle triglycerides, muscles were weighed and lysed in the lysis buffer containing 140 mM NaCl, 50 mM Tris pH 7.4, and 0.1% Triton-X100. A small aliquot of the lysates was used in a triglycerides assay using Stanbio™ Triglycerides LiquiColor™ Reagent.

Gong Y., Liu J., Xue Y., Zhuang Z., Qian S., Zhou W., Li X., Qian J., Ding G, & Sun Z. (2019). Non-monotonic dose-response effects of arsenic on glucose metabolism. Toxicology and applied pharmacology, 377, 114605.

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Western Blot and HDAC3 Assay Protocol

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For western blot, tissues or cells were lysed in RIPA buffer, which contained PMSF and proteinase inhibitor. The concentration of total protein was quantified using Bradford protein assay. Lysates were resolved on SDS-PAGE gel electrophoresis and then transferred to PVDF membranes. The membranes were further treated with skimmed milk and blotted with antibodies of HDAC3 (Abcam 7030), Ampd3 (Abcam 194361), Hsp90 (CST 4874), Tubulin (CST 2148), and OXPHOS proteins (MitoSciences MS604). For HDAC assay, the HDAC3 protein was immunoprecipitated from the muscle protein lysates with 2 mg total protein, followed by a fluorescence-based HDAC assay reaction (Active Motif) performed with the pelleted beads (Sun et al., 2013 (link)).

Song S., Wen Y., Tong H., Loro E., Gong Y., Liu J., Hong S., Li L., Khurana T.S., Chu M, & Sun Z. (2018). The HDAC3 enzymatic activity regulates skeletal muscle fuel metabolism. Journal of Molecular Cell Biology, 11(2), 133-143.

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Protein Expression Analysis in Muscle

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Western blotting was performed on whole-muscle homogenate as previously described (21 (link)) using the following commercially available antibodies: α-tubulin (Ab7291; Abcam), ANT1 (MSA02; MitoSciences), ANT2 (AP1057; Millipore), 4HNE (HNE11-S; Alpha Diagnostic International), OXPHOS (MS604; MitoSciences), COXIV (Invitrogen), CAT (AB1877; Abcam), and SOD2 (AB11889; Abcam), and for protein carbonylation, the OxyBlot Protein Oxidation Detection Kit (S7150; Millipore) was used. Ponceau staining was used to confirm equal loading for antibodies that required the entire membrane (e.g., 4HNE and protein carbonylation). In addition, Western blotting was performed on recovered permeabilized fibers following respiration protocols as previously described (30 (link)). All samples for a given protein were detected on the same membrane using chemiluminescence and the FluorChem HD imaging system (Alpha Innotech, Santa Clara, CA).

Ludzki A., Paglialunga S., Smith B.K., Herbst E.A., Allison M.K., Heigenhauser G.J., Neufer P.D, & Holloway G.P. (2015). Rapid Repression of ADP Transport by Palmitoyl-CoA Is Attenuated by Exercise Training in Humans: A Potential Mechanism to Decrease Oxidative Stress and Improve Skeletal Muscle Insulin Signaling. Diabetes, 64(8), 2769-2779.

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Analyzing Oxidative Phosphorylation Complexes

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Cells were trypsinized, washed two times in ice-cold phosphate-buffered saline (PBS). Cells (5 x 10⁵) were lysed on ice for 20–30 minutes in lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), protease inhibitor cocktail (Complete Mini, Roche Diagnostics Gmbh, Mannheim, Germany), 1% Nonidet P40 (NP-40), 10% glycerol, 50 mM NaF and 1 mM Na₃VO₄. Protein extracts were denatured in loading buffer at room temperature for 20 minutes. Samples were analyzed on 12% SDS-PAGE gels run for 1 hour at 150 V before being transferred to nitrocellulose membranes for 1 hour at 250 mA. Membranes were blocked for 2 hours at room temperature with 5% (w/v) fat-free milk, 0.1% Tween 20 in Tris-buffered saline, pH 7.6, and then incubated over night at 4°C with primary antibodies for oxidative phosphorylation complexes MS604, (Mitosciences, USA) a “cocktail” of antibodies probing complexes 1–5; for complex 1 an antibody against CI subunit NDUFB8, and for complex 2 against SDHB at 1:500 dilutions and for beta-actin at 1:12,000 dilution. Monoclonal mouse anti-beta-actin was used as loading control. Secondary antibody incubations employed a HRP-linked anti-mouse antibody for 1 hour at room temperature. Immunoreactive bands were visualized using chemiluminescence (ECL Western blotting reagent, Pierce, Biotechnology, USA).

Hals I.K., Bruerberg S.G., Ma Z., Scholz H., Björklund A, & Grill V. (2015). Mitochondrial Respiration in Insulin-Producing β-Cells: General Characteristics and Adaptive Effects of Hypoxia. PLoS ONE, 10(9), e0138558.

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