Cytochrome c from equine heart

Manufactured by Merck Group

Sourced in United States, Germany

Cytochrome c from equine heart is a redox-active protein that functions as an essential component of the electron transport chain in mitochondria. It plays a crucial role in the process of cellular respiration, facilitating the transfer of electrons between Complex III and Complex IV of the respiratory chain. This product is purified from the heart tissue of horses and is commonly used in biochemical research and assays related to electron transport and energy metabolism.

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38 protocols using cytochrome c from equine heart

Fabrication of Hybrid Protein-Polymer Materials

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Styrene (STY, >99.5%), divinylbenzene (DVB,
80%), 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS, 98%),
2,2′-azobis(isobutyronitrile) (AIBN, 98%), n-decanol (99%), sodium hydroxide (NaOH), aluminum oxide, lysozyme
from chicken egg white (Lys), carbonic anhydrase from bovine heart
(CA, >90%), cytochrome c from equine heart (CC, >95%), bovine
casein
(Cas), and potassium iodide (KI, >99%) were purchased from Sigma-Aldrich
(St. Louis, Missouri, United States). Ethanol (EtOH), methanol (MeOH),
acetonitrile (ACN), and tetrahydrofuran (THF, >99.8) were purchased
from Biosolve (Valkenswaard, The Netherlands). Hydrochloric acid 37%
(HCl) and glacial acetic acid were obtained from Acros (Geel, Belgium).
Milli-Q water (18.2 MΩcm) was produced by a Sartorius Arium
611UV Ultrapure Water System (Göttingen, Germany). The titanium
devices were purchased from Materialise (Leuven, Belgium), while the
capillary (0.53 mm ID, 0.70 mm OD) was purchased from CMScientific
(Silsden, UK).

Passamonti M., Bremer I.L., Nawada S.H., Currivan S.A., Gargano A.F, & Schoenmakers P.J. (2019). Confinement of Monolithic Stationary Phases in Targeted Regions of 3D-Printed Titanium Devices Using Thermal Polymerization. Analytical Chemistry, 92(3), 2589-2596.

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Chemo-Enzymatic Synthesis of Heparan Sulfate Oligomers

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All chemicals and solvents (ammonium acetate, methanol, water, and formic acid) were of HPLC grade and purchased from Sigma-Alldrich, (St. Louis, MO). Human recombinant FGF 1 expressed in Escherichia coli was a gift from Amgen (Thousand Oaks, CA, USA). Protein calibrants (myoglobin from equine heart, cytochrome c from equine heart, avidin from egg white, concanavalin A from Canavalia ensiformis, and bovine serum albumin) were purchased from Sigma-Aldrich. HS dodecasaccharides were chemo-enzymatically synthesized as previously described [45 (link)]. HS tetrasaccharides were synthesized as previously described based by fluorous supported modular synthesis [46 (link)].

Zhao Y., Singh A., Xu Y., Zong C., Zhang F., Boons G.J., Liu J., Linhardt R.J., Woods R.J, & Amster I.J. (2016). Gas-Phase Analysis of the Complex of Fibroblast Growth Factor 1 with Heparan Sulfate: A Traveling Wave Ion Mobility Spectrometry (TWIMS) and Molecular Modeling Study. Journal of the American Society for Mass Spectrometry, 28(1), 96-109.

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Heparin-binding Protein Characterization

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All chemicals and solvents (ammonium acetate, methanol, water and formic acid) were of HPLC grade and purchased from Sigma-Aldrich. AT III was purchased from Aniara/Hyphen Biomed as lyophilized powder (West Chester, OH). Stock solution of ATIII was made by dissolving the lyophilized protein into HPLC-grade water and then stored at −80 °C. Arixtra was purchased from the hospital formulary and desalted on a BioGel P2 column BioRad (Hercules, CA, USA) before use. Modified Arixtra was chemoenzymatically synthesized as previously described.^{27 (link)} The Hp tetrasaccharide was produced from naturally occurring source as previously described.^{28 (link)} Protein calibrants (myoglobin from equine heart, cytochrome c from equine heart, avidin from egg white, concanavalin A from Canavalia ensiformis and bovine serum albumin) were purchased from Sigma-Aldrich as lyophilized powder.

Zhao Y., Singh A., Li L., Linhardt R.J., Xu Y., Liu J., Woods R.J, & Amster I.J. (2015). Investigating changes in the gas-phase conformation of Antithrombin III upon binding of Arixtra using traveling wave ion mobility spectrometry (TWIMS). The Analyst, 140(20), 6980-6989.

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Lipid-Functionalized Polymer Nanoparticles

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4-Arm, alkylthiol-functionalized PEG (PEG-SH, M_n 20 000 g/mol) and 4-arm, hydroxyl-functionalized PEG (M_n 20 000 g/mol) were purchased from JenKem Technology USA, Inc. (Allen, TX, USA). All lipids were purchased from Avanti Polar Lipids (Alabaster, AL, USA), including: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), anionic 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), and the anionic maleimide-functionalized lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl) butyramide (MPB-PE). 4-Mercaptophenylpropionic acid (4-mercaptohydrocinnamic acid) was purchased from TCI America (Portland, OR, USA). Doxorubicin hydrochloride (DOX) was purchased from Alfa Aesar (Ward Hill, MA, USA). Glutathione (GSH) and cytochrome c from equine heart were purchased from Sigma-Aldrich (Saint Louis, MO, USA). All other reagents and materials were purchased from Fisher Scientific (Pittsburgh, PA, USA) unless otherwise noted. ¹H NMR spectra were acquired under standard quantitative conditions at ambient temperature on a Bruker AV400 NMR spectrometer (Billerica, MA, USA). All samples were dissolved in CDCl₃ (D, 99.8%) + 0.05% V/V TMS.

Liang Y, & Kiick K.L. (2016). Liposome-Cross-Linked Hybrid Hydrogels for Glutathione-Triggered Delivery of Multiple Cargo Molecules. Biomacromolecules, 17(2), 601-614.

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Methylglyoxal-Induced Protein Glycation Assay

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Methylglyoxal (MG) 40% solution, aminoguanidine (AG), bovine serum albumin (BSA), guanidine hydrochloride, L-cysteine, o-phenylenediamine (ο-PD), 5-methylquinoxaline (5-MQ), 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH), 2-deoxy-D-ribose, Cytochrome c from equine heart and 2-thiobarbituric acid (TBA) were obtained from Sigma (St. Louis, MO, USA). 2,4-dinitrophenylhydrazine (DNPH) was purchased from Ajax Finechem (Taren Point, NSW, Australia), whereas L-lysine hydrochloride was obtained from Himedia (L.B.S. Marg, MB, India). Finally, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), trichloroacetic acid and methanol (gradient grade for liquid chromatography) were purchased from Merck (Darmstadt, Germany).

Chayaratanasin P., Adisakwattana S, & Thilavech T. (2021). Protective role of Clitoria ternatea L. flower extract on methylglyoxal-induced protein glycation and oxidative damage to DNA. BMC Complementary Medicine and Therapies, 21, 80.

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Protein Mixture Preparation for Mass Spectrometry

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Ubiquitin from bovine erythrocytes, cytochrome c from equine heart, and myoglobin from equine heart were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trimethylamine was purchased from EMD Chemicals (Gibbstown, NJ, USA). HPLC-grade methanol and Optima LC/MS-grade water were purchased from Fisher Scientific (Fair Lawn, NJ, USA), and acetic acid was purchased from Mallinckrodt (Phillipsburg, NJ, USA). Protein stock solutions were prepared at a concentration of approximately 1 mg/mL. Stocks were diluted to a concentration of 1 μM, 2 μM, and 2 μM for ubiquitin, cytochrome c, and myoglobin, respectively, in 49.5/49.5/1, by volume, water/methanol/acetic acid. A simple protein mixture of 1μM cytochrome c and 1 μM myoglobin was prepared in 49.5/49.5/1, by volume, water/methanol/acetic acid.

Foreman D.J., Bhanot J., Lee K.W, & McLuckey S.A. (2020). Valet Parking for Protein Ion Charge State Concentration: Ion/Molecule Reactions in Linear Ion Traps. Analytical chemistry, 92(7), 5419-5425.

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Native Mitochondrial Protein Complex Analysis

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Isolated mitochondria were resuspended in 1.5 M aminocaproic acid, 50 mM Bis‐Tris/HCl pH 7.0. The samples were solubilized with 4 mg of n‐dodecyl‐β‐D‐maltoside (Sigma) per mg of protein. After 5 min of incubation on ice, samples were centrifuged at 18,000 × g at 4°C for 30 min. The supernatant was collected and resuspended with Sample Buffer (750 mM aminocaproic acid, 50 mM Bis‐Tris/HCl pH 7.0, 0.5 mM EDTA and 5% Serva Blue G). Native samples were separated using NativePAGE 3–12% Bis‐Tris gels (Thermo Fisher Scientific) according to the manufacturer’s protocol.
For the detection of the activity of mitochondrial respiratory chain complexes, gels were stained with the following solutions. Complex II (succinate dehydrogenase): 5 mM Tris–HCl pH 7.4, 0.2 mM phenazine methosulfate (Sigma), 20 mM succinate, and 1 mg/ml nitrotetrazolium blue chloride; Complex IV (cytochrome c oxidase): 50 mM potassium phosphate pH 7.4, 1 mg/ml 3′,3′‐diaminobenzidine tetrahydrochloride hydrate (Sigma), 24 units/ml catalase from bovine liver (Sigma), 1 mg/ml cytochrome c from equine heart (Sigma), and 75 mg/ml sucrose.

Brischigliaro M., Cabrera‐Orefice A., Sturlese M., Elurbe D.M., Frigo E., Fernandez‐Vizarra E., Moro S., Huynen M.A., Arnold S., Viscomi C, & Zeviani M. (2022). CG7630 is the Drosophila melanogaster homolog of the cytochrome c oxidase subunit COX7B. EMBO Reports, 23(8), e54825.

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Biopolymer-Based Cytochrome c Delivery

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Cytochrome c from equine heart, reduced glutathione ethyl ester, and a protease inhibitor cocktail were from Sigma-Aldrich (St. Louis, MO). Acetonitrile (HPLC grade) was from Fisher (Waltham, MA). Succinimidyl-3-(2-pyridyldithio) propionate (SPDP) was from Proteochem (Denver, CO). Poly (lactide-co-glycolide)-SH with a thiol end cap (PLGA-SH, 30,000 Da, copolymer ratio 1:1) was from Akina, Inc (West Lafayette, IN). 4’, 6-Diamidino-2-phenylindole (DAPI) and propidium iodide (PI) were purchased from Invitrogen (Grand Island, NY). All the reagents were used without further purification. All other chemicals were from various commercial suppliers and were at least of analytical grade. HeLa cells were purchased from the American Type Culture Collection (Manassas, VA) and grown according to the vendor’s instruction.

Morales-Cruz M., Figueroa C.M., González-Robles T., Delgado Y., Molina A., Méndez J., Morales M, & Griebenow K. (2014). Activation of caspase-dependent apoptosis by intracellular delivery of cytochrome c-based nanoparticles. Journal of Nanobiotechnology, 12, 33.

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Chemo-enzymatic Synthesis of HS Oligosaccharides

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All chemicals and solvents (ammonium acetate, methanol, water, and formic acid) were of HPLC grade and purchased from Sigma-Aldrich, (St. Louis, MO). Protein calibrants (myoglobin from equine heart, cytochrome c from equine heart, avidin from egg white, concanavalin A from Canavalia ensiformis, and bovine serum albumin) were purchased from Sigma-Aldrich. HS hexasaccharides (dp6) to dodecasaccharides (dp12) were chemo-enzymatically synthesized as previously described [34 (link)]. HS tetrasaccharide (dp4) was chemically synthesized as previously described by fluorous supported modular synthesis [35 (link)].

Zhao Y., Yang J.Y., Thieker D.F., Xu Y., Zong C., Boons G.J., Liu J., Woods R.J., Moremen K.W, & Amster I.J. (2018). A TRAVELING WAVE ION MOBILITY SPECTROMETRY (TWIMS) STUDY OF THE ROBO1-HEPARAN SULFATE INTERACTION. Journal of the American Society for Mass Spectrometry, 29(6), 1153-1165.

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Enzymatic Synthesis of L-Ascorbic Acid

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L-Gulono-1,4-lactone, D-gluconic acid-γ-lactone, cytochrome C from
equine heart, flavin adenine dinucleotide (FAD), reduced glutathione,
metaphosphoric acid and ascorbate oxidase were purchased from Sigma-Aldrich (St.
Louis, MO, USA). Dithiothrietol (DTT), 2-(N-morpholino) ethane sulfonic acid
(MES), Tris, NaCl, glycine, sodium dodecyl sulfate (SDS), and glycerol were
purchased from Fisher Scientific (Fair Lawn, NJ, USA).
Ethylenediaminetetraacetic acid (EDTA) was acquired from Promega (Madison, WI,
USA). D-Galactono-1,4-lactone was purchased from Carbomer (San Diego, CA, USA).
L-Galactono-1,4-lactone was from Santa Cruz Biotechnology (Dallas, TX, USA) and
L-galactose was from TCI America (Portland, OR, USA).

Aboobucker S.I., Suza W.P, & Lorence A. (2017). Characterization of Two Arabidopsis L-Gulono-1,4-lactone Oxidases, AtGulLO3 and AtGulLO5, Involved in Ascorbate Biosynthesis. Reactive oxygen species (Apex, N.C.), 4(12), 389-417.

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