Anti cytochrome c antibody

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-cytochrome c antibody is a laboratory reagent used for the detection and quantification of cytochrome c, a key component of the mitochondrial electron transport chain. This antibody can be employed in various analytical techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to study the expression and localization of cytochrome c in biological samples.

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Cytochrome c antibody Anti-cytochrome c Cytochrome c

10 protocols using anti cytochrome c antibody

Investigating Zinc-Induced TR3 and Cytochrome c Dynamics

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Both PC-3 and LNCaP cells were cultured and treated with zinc as required, and immunofluorescence staining was performed. Briefly, after being washed with PBS, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature. To identify TR3 protein expression, cells were first incubated with an anti-TR3 immunoglobin G (IgG) antibody (sc365113; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and then with a corresponding FITC-conjugated anti-IgG antibody (AS10115; Agrisera, Vasterbotten, Sweden) as the secondary antibody. To visualize nuclei, cells were stained with 50 µg/mL of 4,6-diamidino-2-phenylindole (B28718; Sigma-Aldrich) containing 100 mg/mL of Dnase-free Rnase A. To visualize mitochondria, cells were incubated with an anti-HSP60 mouse IgG antibody (sc136291; Santa Cruz Biotechnology, Inc.) and reacted with a Texas red-conjugated anti–mouse IgG (sc69786; Santa Cruz Biotechnology, Inc.) as the secondary antibody.
Additionally, to display the translocation of cytochrome c in PC-3 and LNCaP cells, the experimental procedure was performed as described by the manufacturer’s protocol. Cells were incubated with an anti-cytochrome c antibody (sc13156; Santa Cruz Biotechnology, Inc.) and then the FITC-conjugated antibody. Immunofluorescence staining procedures for both nuclei and mitochondria were then conducted.

Ma D., Guo Y., Fu Y., Wu J, & Ren R. (2023). Temporal relationship of the orphan receptor TR3 translocation and expression with zinc-induced apoptosis in prostate cancer cells. Translational Andrology and Urology, 12(3), 444-454.

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Cytochrome c Localization in Cells

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Cells in each treatment group were incubated with 0.5 mM MTR (excitation wavelength 579 nm, emission wavelength 599 nm) in phosphate-buffered saline at 37°C for 20 minutes, fixed in 4% paraformaldehyde at 4°C for 15 minutes, and subsequently permeabilized with 0.1% Triton X-100 at 4°C for 5 minutes; between steps, cells were washed with phosphate-buffered saline. After permeation, cells were blocked with goat serum at 37°C for one hour, and then incubated with anti-cytochrome c antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. After being washed with phosphate-buffered saline, the samples were incubated with fluorescein isothiocyanate-conjugated secondary antibody. Cells were also labeled with the nucleus dye, Hoechst 33258, and observed by inverted confocal laser scanning microscopy.

Wang H., Wang X., Zhang S., Wang P., Zhang K, & Liu Q. (2014). Sinoporphyrin sodium, a novel sensitizer, triggers mitochondrial-dependent apoptosis in ECA-109 cells via production of reactive oxygen species. International Journal of Nanomedicine, 9, 3077-3090.

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Mitochondrial Cytochrome c Localization

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After the appropriate treatment, the cells were washed with PBS and stained with 200 nM MitoTracker Red CMXRos dye (Molecular Probes, Inc., France), followed by incubation for 30 min at room temperature, to visualize the mitochondria. The cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked using 3% serum dissolved in PBS for 30 min at room temperature. The cells were then probed with anti-cytochrome c antibody (1 : 40; Santa Cruz Biotechnology) overnight at 4°C. The cells were washed with PBS twice and incubated with FITC-conjugated secondary antibody (1 : 40; Biovision, China) for 2 h in the dark at 37°C. After washing, images of stained cells were obtained using a fluorescence microscope.

Wang L., Huang H., Fan Y., Kong B., Hu H., Hu K., Guo J., Mei Y, & Liu W.L. (2014). Effects of Downregulation of MicroRNA-181a on H2O2-Induced H9c2 Cell Apoptosis via the Mitochondrial Apoptotic Pathway. Oxidative Medicine and Cellular Longevity, 2014, 960362.

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Cytochrome c and Mitochondria Staining in Pancreatic Cancer Cells

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For cytochrome c and mitochondria staining, PANC-1, AsPC-1, and Panc 02 cells were seeded on ϕ12-mm round glass cover-slips (2 × 10⁵ cells per cover-slip) and treated with DMSO or OG for 24 h. Cells were pre-incubated with 200 nM of MitoTracker^® Orange CMTMRos for 30 min at 37 °C before harvest. Cells were fixed with 3.7% paraformaldehyde and blocked with 3% BSA in PBS for 30 min. Anti-cytochrome c antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-13156; 1:100) was applied for 1 h at room temperature, followed by incubation with Alexa Fluor 488 Dye (1:500) for another 1 h at room temperature. Nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI) for 5 min at room temperature. Images were observed and analyzed using Leica TCS SP5 II confocal microscope and LAS AF Lite 4.0 software (Leica, Wetzlar, Germany).

Chua K.V., Fan C.S., Chen C.C., Chen L.L., Hsieh S.C, & Huang T.S. (2019). Octyl Gallate Induces Pancreatic Ductal Adenocarcinoma Cell Apoptosis and Suppresses Endothelial-Mesenchymal Transition-Promoted M2-Macrophages, HSP90α Secretion, and Tumor Growth. Cells, 9(1), 91.

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Mitochondrial Apoptosis Assessment in Transgenic Mice

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The mitochondrial apoptosis pathway was assessed by measuring caspase activity, cytochrome c release from the mitochondria, and bcl-2 levels in the liver obtained from young (6–7 months old) Tg(TXN)^+/0 and WT mice. The caspase activities, cytochrome c release, and bcl-2 levels were determined by Western blot assay. The caspase-3 activity in liver tissue were visualized by the cleavage forms of caspase-3 (p20) as an indicator of caspase activity using anti-caspase-3 antibody (Cell Signaling Technology, Inc., Danvers, MA). The intensities of cleavage bands corresponding to p20 for caspase-3 were quantified by densitometry using ImageQuant v5.0, and β-actin was used as a loading control. The cytochrome c release from mitochondria was measured as levels of cytochrome c in the cytosolic fractions from liver. Equal amounts of protein were separated on a 4–20% SDS-polyacrylamide gel, transferred to nitrocellulose membranes, and subjected to Western blotting with an anti-cytochrome c antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The bcl-2 levels were also measured by Western blot using anti-bcl-2 antibody (Cell Signaling Technology, Inc., Danvers, MA). Data were normalized to β-actin loading controls. The data were expressed as relative units respective to β-actin.

Flores L.C., Roman M.G., Cunningham G.M., Cheng C., Dube S., Allen C., Van Remmen H., Hubbard G.B., Saunders T.L, & Ikeno Y. (2018). Continuous overexpression of thioredoxin 1 enhances cancer development and does not extend maximum lifespan in male C57BL/6 mice. Pathobiology of Aging & Age Related Diseases, 8(1), 1533754.

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Mitochondrial Dynamics in MIAPaCa-2 Cells

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MIAPaCa-2 cells were plated on 18-mm glass coverslips and grown to approximately 70% confluence. The cells were treated with sorafenib and melatonin, alone and in combination for 12 h, then permeabilized, and incubated with 500 nM MitoTracker Red probe (Molecular Probes Inc., Eugene, OR, USA) for 30 min at 37°C. Next, the cells were washed twice with PBS, fixed in an acetic acid:ethanol (1:2) solution for 10 min at -20°C, and then incubated overnight at 4°C with an anti-cytochrome c antibody (1:50; Santa Cruz Biotechnologies, Dallas, TX, USA). On the following day, the cells were washed twice with PBS and incubated with fluorescence-labeled anti-mouse secondary antibody (1:100; Dianova, Hamburg, Germany) for 1 h at room temperature. The cells were also stained with DAPI to visualize nuclei. The slides were then washed twice with PBS, mounted with DABCO (Sigma-Aldrich, St. Louis, MO, USA), and viewed under a confocal laser-scanning microscope (Fluo View 1000; Olympus).

Fang Z., Jung K.H., Yan H.H., Kim S.J., Rumman M., Park J.H., Han B., Lee J.E., Kang Y.W., Lim J.H, & Hong S.S. (2018). Melatonin Synergizes with Sorafenib to Suppress Pancreatic Cancer via Melatonin Receptor and PDGFR-β/STAT3 Pathway. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 47(5).

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Measuring Mitochondrial Cytochrome C Release

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The amount of cytochrome C released from the mitochondrial intermembrane space into the cytosol was determined by a western blot analysis. After washing with ice-cold phosphate-buffered saline, cells were resuspended in permeabilization buffer containing 75 mM NaCl, 1 mM NaH₂PO₄, 8 mM Na₂PO₄, 250 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, additional protease inhibitors, and 0.05% digitonin. Following centrifugation at 15000 rpm at 4℃ for 10 min, the supernatant was separated from the pellet consisting of mitochondria and cellular debris. Equal amounts of protein were loaded on a 15% acrylamide gel and separated by SDS-PAGE. Nitrocellulose paper was then blocked with 0.5% nonfat dry milk, probed with 0.5 µg/mL polyclonal anti-cytochrome C antibodies (1 : 200, Santa Cruz Biotechnology, Dalas, TX, USA), then with secondary antibodies (rabbit) diluted 1 : 5000. Immunoblots were developed with ECL-plus reagents from Amersham according to the manufacturer's instructions.

Sung S.K., Woo J.S., Kim Y.H., Son D.W., Lee S.W, & Song G.S. (2016). Sildenafil Ameliorates Advanced Glycation End Products-Induced Mitochondrial Dysfunction in HT-22 Hippocampal Neuronal Cells. Journal of Korean Neurosurgical Society, 59(3), 259-268.

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Cytochrome c Fractionation and Detection

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The cytosolic fractions were prepared and analyzed by western blots using anti-cytochrome c antibodies (Santa Cruz) as described [12 (link)].

Dalal S., Zha Q., Singh M, & Singh K. (2016). Osteopontin-stimulated apoptosis in cardiac myocytes involves oxidative stress and mitochondrial death pathway: role of a pro-apoptotic protein BIK. Molecular and cellular biochemistry, 418(1-2), 1-11.

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Cladoloside C2 Apoptosis Induction

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Cladoloside C₂ was isolated and purified following the procedure published by Silchenko et al. [28 (link)], and was dissolved in sterilized distilled water. Annexin V was obtained from BD Biosciences Clontech (Palo Alto, CA, USA). Anti-Fas, anti-procaspase 8, anti-procaspase-3, anti-procaspase 9, and anti-cytochrome c antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against poly (ADP-ribose) polymerase (PARP), JNK, and p-JNK were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-β-actin antibody was obtained from Sigma (St. Louis, MO, USA). Unless otherwise stated, all other chemicals were purchased from Sigma.

Yun S.H., Sim E.H., Han S.H., Kim T.R., Ju M.H., Han J.Y., Jeong J.S., Kim S.H., Silchenko A.S., Stonik V.A, & Park J.I. (2017). In vitro and in vivo anti-leukemic effects of cladoloside C2 are mediated by activation of Fas/ceramide synthase 6/p38 kinase/c-Jun NH2-terminal kinase/caspase-8. Oncotarget, 9(1), 495-511.

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Cytochrome c Detection in Cytosol

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The cytosolic fractions were prepared and analyzed by western blots using anti-cytochrome c antibodies (Santa Cruz) as described [29 (link)].

Dalal S., Connelly B., Singh M, & Singh K. (2018). NF2 signaling pathway plays a pro-apoptotic role in β-adrenergic receptor stimulated cardiac myocyte apoptosis. PLoS ONE, 13(4), e0196626.

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