Anti cytochrome c

Manufactured by Santa Cruz Biotechnology

Sourced in United States, Germany

Anti-cytochrome c is a laboratory reagent used in various biochemical and cell biology applications. It is an antibody that specifically binds to and detects the presence of cytochrome c, a crucial protein involved in cellular respiration and apoptosis (programmed cell death) pathways. The primary function of Anti-cytochrome c is to serve as a detection and analytical tool for researchers studying mitochondrial function, cell signaling, and cell death mechanisms.

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Lab products found in correlation

53 protocols using anti cytochrome c

HUVEC Cell Culture and Treatments

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Human umbilical vein endothelial cells (HUVECs) were obtained from ATCC. HUVECs were cultured with M199 basal medium supplemented with low-serum growth supplement and penicillin (50 IU/ml)-streptomycin (50 μg/ml). trypsin-EDTA was used to passage cells. M199 and trypsin-EDTA were obtained from Gibco (Grand Island, NY, USA). Low-serum growth supplement was purchased from Cascade (Portland, OR, USA). Additionally. DHE, L-NIO, SNP, 1400W, penicillin and streptomycin were all purchased from Sigma (St. Louis, MO, USA). Anti-β-actin, anti-eNOS, anti-iNOS, anti-cytochrome c, anti-P53, anti-p-P53, anti- nitrotyrosine, anti-Bcl-2,anti-Bax were all obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Baicalein was obtained from Sigma and solved in DMSO.

Chan S.H., Hung C.H., Shih J.Y., Chu P.M., Cheng Y.H., Tsai Y.J., Lin H.C, & Tsai K.L. (2016). Baicalein is an available anti-atherosclerotic compound through modulation of nitric oxide-related mechanism under oxLDL exposure. Oncotarget, 7(28), 42881-42891.

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Retinal Protein Expression Analysis

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The neural retina was detached from the RPE, and both tissues were homogenized as previously described [13 (link), 14 (link), 34 (link)]. Proteins (50 μg/sample) were separated in SDS, 12% polyacrylamide gel, transferred to polyvinylidene difluoride membranes, and incubated overnight at 4 °C with a mouse monoclonal anti-cytochrome c (1:1000, Santa Cruz Biotechnology, Dallas TX, USA), a rabbit polyclonal anti-TOM20 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), a rabbit polyclonal anti-VDAC (1:300, Santa Cruz Biotechnology, Dallas, TX, USA), a rabbit polyclonal anti-BDNF (1:500, Alomone Labs, Jerusalem, Israel), a mouse polyclonal anti-RPE65 (1:500; EMD Millipore, Darmstadt, Germany, MAB5428) and a mouse anti-β-actin (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA). The following secondary antibodies were used: a donkey anti-mouse (1:2000, Jackson Laboratory, Bar Harbor, ME, USA) and a donkey anti-rabbit (1:2000, Jackson Laboratory, Bar Harbor, ME, USA). Densitometric signals were quantified using ImageQuant software and adjusted by the density of β-actin. For each group, the mean of five homogenates were averaged and taken as the representative value.

Dieguez H.H., Calanni J.S., Romeo H.E., Alaimo A., González Fleitas M.F., Iaquinandi A., Chianelli M.S., Keller Sarmiento M.I., Sande P.H., Rosenstein R.E, & Dorfman D. (2021). Enriched environment and visual stimuli protect the retinal pigment epithelium and photoreceptors in a mouse model of non-exudative age-related macular degeneration. Cell Death & Disease, 12(12), 1128.

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Betulinic Acid Modulates Prostate Cancer

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The androgen-responsive human prostate cancer LNCaP cells and the androgen-refractory DU145 cells were maintained in RPMI media purchased from HyClone Laboratories (Logan, UT, USA). Betulinic acid (≥98% purity) was obtained from A. G. Scientific, Inc. (San Diego, CA, USA). Anti-p53, anti-Ser15-p53, anti-p21/Waf1, anti-NF-κB/p65, anti-IκBα, anti-p-IκBα, anti-p-IKKα, anti-Bax, anti-Bcl-2, anti-cytochrome C and anti-PARP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Caspase 9 and Caspase 3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Propidium iodide was obtained from EMD Millipore (Billerica, MA, USA). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The Cell Death Detection ELISA^PLUS kit was obtained from Roche Diagnostics (Mannheim, Germany).

Shankar E., Zhang A., Franco D, & Gupta S. (2017). Betulinic Acid-Mediated Apoptosis in Human Prostate Cancer Cells Involves p53 and Nuclear Factor-Kappa B (NF-κB) Pathways. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 22(2), 264.

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Molecular Mechanisms of Oxidative Stress

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Anti-GAPDH, anti-phospho-JNK1/2, anti-β-actin, anti-caspase-3, anti-caspase-7, anti-caspase-9, anti-poly(ADP-ribose) polymerase (PARP), anti-Bax, anti-Bad, anti-Bcl-2, anti-cytochrome c, anti-Akt, and anti-COX IV antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). SP600125, Z-VAD-FMK, MitoTEMPO, surfactin, and urban PM (SRM 1648a) were purchased from Sigma (St. Louis, MO, USA). N-acetyl-L-cysteine (NAC) and diphenyleneiodonium chloride (DPI) were taken from Biomol (Plymouth Meeting, PA).

Vo T.T., Liu J.F., Wu C.Z., Lin W.N., Chen Y.L, & Lee I.T. (2020). Surfactin from Bacillus subtilis induces apoptosis in human oral squamous cell carcinoma through ROS-regulated mitochondrial pathway. Journal of Cancer, 11(24), 7253-7263.

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Evaluating Mitochondrial Cytochrome C Localization

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Ins-1 cells cultured on coverslips were incubated with a vehicle (control, DMSO) or 50 µM BPCA for 1 h, after which 0.2 mM PA was added. After 24 h, the coverslips were washed twice with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. The fixed cells were washed with PBS, blocked with PBS containing 1% BSA and 0.1% Triton X-100 for 30 min at room temperature, and incubated overnight with anti-cytochrome C (Santa Cruz) and anti-Tom 20 (Santa Cruz) at 4 °C. Cells were then stained with fluorescence-conjugated secondary antibody (Life Technologies, Waltham, MA, USA) for 2 h, mounted using VECTASHIELD (Vector Laboratories, Burlingame, CA, USA), and observed under a confocal microscope (LSM700, Zeiss, Oberkochen, Germany). To evaluate cytochrome C and Tom 20, five random fields were selected in each experiment and 10–12 cells were imaged in each field. To quantitatively evaluate the fluorescent images, the RGB image was analyzed using the ImageJ software (NIH), and the mean value was shown in bar graphs.

Cha S.H., Zhang C., Heo S.J, & Jun H.S. (2021). 5-Bromoprotocatechualdehyde Combats against Palmitate Toxicity by Inhibiting Parkin Degradation and Reducing ROS-Induced Mitochondrial Damage in Pancreatic β-Cells. Antioxidants, 10(2), 264.

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Cytoplasmic Cytochrome-c Extraction from Cells

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SK-MEL-28 and Mia-PaCa-2 cells were treated with Amblyomin-X. Cells were harvested and were washed twice in PBS. For cytoplasmic cytochrome-c measurement, a modified extraction procedure was used compared to the one we employed for the analysis of GRP78, GADD153, and PARP. Briefly, to obtain a cytoplasmic proteins fraction, cells were re-suspended in relatively weak lysis buffer (HEPES–KOH 10 mM; pH 7.4, EDTA 0.1 mM; bis‐etilenoglicol (β‐aminoethyl ether); EGTA 1 mM; sucrose 250 mM; orthovanadate (Na3VO4) 1 mM, PMSF 1.0 mM, and dithiothreitol (DTT) 10 mM) and were incubated on ice for 15 min. Samples were then centrifuged at 10,000 g for 15 min at 4 °C to sediment unbroken cells, nuclei, and the heavy membrane fractions containing mitochondria or other cellular compartments. Supernatants containing mostly cytoplasmic proteins were collected and were subjected to immunoblot analysis using anti-cytochrome-c (Santa Cruz Biotechnology, Inc., USA).

Morais K.L., Pacheco M.T., Berra C.M., Bosch R.V., Sciani J.M., Chammas R., de Freitas Saito R., Iqbal A, & Chudzinski-Tavassi A.M. (2016). Amblyomin-X induces ER stress, mitochondrial dysfunction, and caspase activation in human melanoma and pancreatic tumor cell. Molecular and Cellular Biochemistry, 415, 119-131.

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Western Blot Analysis of Apoptosis Signaling

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Total protein was extracted from cell samples by using RIPA buffer (Beyotime). 50 μg of the extracted and the co-precipitated proteins were separated by 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking in 5% nonfat milk for 2 h at room temperature, the membranes were incubated with anti-p-Bad (1:200, Santa Cruz), anti-p-AKT (1:200, Santa Cruz), anti-PTEN (1:200, Santa Cruz), anti-Bad (1:200, Santa Cruz), anti-AKT (1:200, Santa Cruz), anti-Bcl-2 (1:200, Santa Cruz), anti-cytochrome c (1:200, Santa Cruz), anti-caspase-9 (1:200, Santa Cruz) and anti-caspase-3 (1:200, Santa Cruz). After incubation with primary antibodies, membranes were washed and incubated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz) for 2 h at room temperature. Proteins on the membrane were visualized by using an enhanced chemiluminescence detection kit (Pierce, Rockford, IL, USA). Glyceraldehyde-3-phosphate (GAPDH) was used as an internal control to ensure equal protein loading. Mitochondria/Cytosol Fraction Kit (BioVision, Milpitas, CA, USA) was used to separate the mitochondria fraction and cytosol fraction before detection of cytochrome c.

Ye J., Sun D., Yu Y, & Yu J. (2020). Osthole resensitizes CD133+ hepatocellular carcinoma cells to cisplatin treatment via PTEN/AKT pathway. Aging (Albany NY), 12(14), 14406-14417.

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Western Blot Analysis of Signaling Proteins

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Western blot analysis was performed as described previously [34 (link)]. The following antibodies were used in this study: anti-AKT (Cell Signaling, 2920S), anti-phospho-ERK(CellSignaling,4370S), anti-phospho-AKT(Ser473) (Epitomics,2118-1), anti-GAPDH(Santa Cruz Biotechnology, SC-32233), PARP(Santa Cruz Biotechnology, SC-8007), anti-caspase-3(Cell Signaling, 9665S), anti-actin(Sigma, A4700), anti-cytochrome C(Santa Cruz Biotechnology, SC-13156), anti-Tom20(Santa Cruz Biotechnology, SC-136211), anti-Mcl-1(Santa Cruz Biotechnology, SC-819), anti-GFP(Sigma, G1544), anti-ERK(Cell Signaling, 4695S), anti-TNFAIP1(proteintech, 60327), anti-LIN28(proteintech,11724), anti-GBP1(proteintech, 15303), anti-Bcl-2(proteintech, 12789), anti-Bxl-xl(proteintech, 26967), anti-GSK3α(Cell Signaling, 9338), anti-phospho-GSK3α(ser21)(Boster, P03152), anti-GSK3β(Cell Signaling, 12456), anti-phospho-GSK3β(ser9)( Cell Signaling, 5558).

Shuang W., Hou L., Zhu Y., Li Q, & Hu W. (2017). Mcl-1 stabilization confers resistance to taxol in human gastric cancer. Oncotarget, 8(47), 82981-82990.

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Western Blot Analysis of Apoptosis Markers

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Western blot analysis was performed as previously described52 (link)53 (link). The cells were lysed with RIPA buffer containing protease and phosphatase inhibitors. After centrifugation, the supernatant was collected and quantified. The proteins were then separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% non-fat milk, the membranes were probed with anti-TRPC6 (Abcam, Cambridge, MA, USA), anti-cytochrome c (Santa Cruz Biotechnology, Dallas, TX, USA), anti-caspase-3 (Cell Signaling Technology, Danvers, MA, USA), and anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), followed by incubation with a florescence-labeled secondary antibody. Western blot bands were scanned using the Odyssey Imaging System (LI-COR, Lincoln, NE, USA). GAPDH (Zhongshanjinqiao, Inc., Beijing, China) was used as internal control.

Zhang Y., Qin W., Zhang L., Wu X., Du N., Hu Y., Li X., Shen N., Xiao D., Zhang H., Li Z., Zhang Y., Yang H., Gao F., Du Z., Xu C, & Yang B. (2015). MicroRNA-26a prevents endothelial cell apoptosis by directly targeting TRPC6 in the setting of atherosclerosis. Scientific Reports, 5, 9401.

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Protein Immunoblotting Assay Protocol

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All chemicals were purchased from Sigma (St. Louis, MO, USA). For immunoblotting, the following antibodies were used: anti-ANT2, anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), anti-Tom20, anti-cytochrome C, anti-53BP1 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-ATP5B (Sigma, St. Louis, MO, USA), anti-γH2AX (Millipore, Billerica, MA, USA), anti-NDUFA9, anti-SDHA, anti-Cox5a, anti-UQCRC2, anti-ANT1 and anti-VDAC (Abcam, Cambridge, UK). All antibodies were diluted 1:1000 in 2.5% non-fat milk. Horseradish peroxidase (HRP) conjugated β-actin (ThermoFisher, Waltham, MA, USA) was used as a loading control. IgG-HRP anti-rabbit (170-6515) and anti-mouse (170-6516) secondary antibodies produced in goat were purchased from BioRad Laboratories (Hercules, CA, USA). Secondary antibodies were diluted 1:10,000 in 2.5% non-fat milk.

Hubackova S., Davidova E., Rohlenova K., Stursa J., Werner L., Andera L., Dong L., Terp M.G., Hodny Z., Ditzel H.J., Rohlena J, & Neuzil J. (2018). Selective elimination of senescent cells by mitochondrial targeting is regulated by ANT2. Cell Death and Differentiation, 26(2), 276-290.

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