All reagents were analytical grade. Ultrapure water (18.2 MΩ·cm) from a Mill-Q system (Merck Millipore, Darmstadt, Germany) was used throughout. Laboratory containers were rinsed with H2O prior to use. Standard solutions of all metals (lead(II) nitrate, cadmium(II) nitrate tetrahydrate, potassium dichromate(VI), iron(III) chloride hexahydrate, nickel(II) sulfate hexahydrate, copper(II) sulfate pentahydrate, manganese(II) chloride tetrahydrate, magnesium(II) chloride hexahydrate, iron(II) sulfate heptahydrate, aluminum(III) sulfate hydrate, barium(II) chloride, vanadium(III) chloride, and cobalt(II) sulfate pentahydrate) were purchased from Sigma-Aldrich (St. Louis, MO, USA). L-ascorbic acid (97%), L-cysteine, Tris base (99.9%), bathophenanthroline (97%), dimethylglyoxime (99%), dithiooxamide (98.5%), and sodium fluoride were purchased from Sigma-Aldrich. Sodium acetate, ammonium acetate, hydrochloric acid, sodium hydroxide, and glacial acetic acid were obtained from Fisher Scientific (Pittsburgh, PA, USA). Whatman (grade 1) filter paper was purchased from Apollo Presentation Products (Booneville, MS, USA).
Bathophenanthroline
Manufactured by Merck Group
Sourced in United States
Bathophenanthroline is a chemical compound used as a laboratory reagent. It is a bidentate ligand that forms a stable coordination complex with various metal ions, including iron, copper, and nickel. Bathophenanthroline is commonly used in analytical techniques, such as spectrophotometry, to detect and quantify the presence of these metal ions.
Automatically generated - may contain errors
Lab products found in correlation
L ascorbic acid, by Merck Group (2 mentions)
Copper 2 sulfate pentahydrate, by Merck Group (2 mentions)
Dimethylglyoxime, by Merck Group (1 mentions)
Dithiooxamide, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Thermo Fisher Scientific (1 mentions)
Nickel 2 sulfate hexahydrate, by Merck Group (1 mentions)
Manganese 2 chloride tetrahydrate, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Thermo Fisher Scientific (1 mentions)
Ammonium acetate, by Thermo Fisher Scientific (1 mentions)
Sodium acetate, by Thermo Fisher Scientific (1 mentions)
Mill q system, by Merck Group (1 mentions)
Lead 2 nitrate, by Merck Group (1 mentions)
Cadmium 2 nitrate tetrahydrate, by Merck Group (1 mentions)
Magnesium 2 chloride hexahydrate, by Merck Group (1 mentions)
Aluminum 3 sulfate hydrate, by Merck Group (1 mentions)
Glacial acetic acid, by Thermo Fisher Scientific (1 mentions)
Vanadium 3 chloride, by Merck Group (1 mentions)
Tris base, by Merck Group (1 mentions)
L cysteine, by Merck Group (1 mentions)
Iron 3 chloride hexahydrate, by Merck Group (1 mentions)
6 protocols using bathophenanthroline
1
Determination of Heavy Metal Ions
2
Chemical Reagents for Analytical Protocols
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All chemicals used in the analysis, such as sodium tripolyphosphate, sodium erythorbate, sodium nitrite, bathophenanthroline, potassium iodide, and cholroform, were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA).
3
Quantifying Serum and Liver Iron
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Serum iron levels were quantified using the MAK025 iron assay kit (Sigma-Aldrich), following the manufacturer's protocol.
To quantify liver iron levels, the livers were first digested at 65°C in an acid digest solution [3 M HCl (Sigma-Aldrich), 10% trichloroacetic acid (Sigma-Aldrich)] overnight, with 1 ml of acid digest for every 100 mg of tissue. After digestion, the samples were vortexed and centrifuged at 830g for 5 min, and then 20 µl of the supernatant was added to 1 ml of chromogen reagent [2.25 M sodium acetate (Sigma-Aldrich) pretreated with Chelex 100 (Bio-Rad), 0.01% bathophenanthroline (Sigma-Aldrich), 0.1% thioglycolic acid (Sigma-Aldrich)]. The absorbances were read at 535 nm using a spectrometer (Beckman DU-640, Beckman Coulter) and the iron levels were calculated by comparing the absorbances with a serial dilution of iron standard (Sigma-Aldrich) mixed with the chromogen reagent.
To quantify liver iron levels, the livers were first digested at 65°C in an acid digest solution [3 M HCl (Sigma-Aldrich), 10% trichloroacetic acid (Sigma-Aldrich)] overnight, with 1 ml of acid digest for every 100 mg of tissue. After digestion, the samples were vortexed and centrifuged at 830
4
Nanoparticle Fabrication and Characterization
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1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine
(DOPE: 18:1 (Δ9-Cis) was purchased from Avanti Polar Lipids
(Alabaster, AL). N-(3-(Dimethylamino)propyl)-N′-ethylcarbodiimide (EDC), N-hydroxysuccinimide
(NHS), 4-pentynoic acid, copper(II) sulfate pentahydrate,l -ascorbic acid, bathophenanthroline, Triton X-100, and sodium bicarbonate
were obtained from Sigma–Aldrich Chemical Co. (St. Louis, MO).
BCA Protein Assay Reagent and HPLC grade methanol and water were purchased
from Fisher Scientific (Pittsburgh, PA). Mouse mammary carcinoma (4T1)
cells were acquired from ATCC (Manassas, VA). Roswell Park Memorial
Institute (RPMI) medium 1640, 1× phosphate buffered saline (PBS),
fetal bovine serum (FBS), trypsin, and penicillin–streptomycin
were all purchased from Mediatech, Inc. (Manassas, VA). Plasmocin
was obtained from InvivoGen (San Diego, CA). Hoechst 33342 nucleic
acid stain, and DID (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine
perchlorate) was obtained from Life Technologies (Carlsbad, CA). Azide-Fluor
545 was purchased from KeraFAST, Inc. (Boston, MA). Formalin was purchased
from JT Baker (Center Valley, PA). Sepharose CL-4B was purchased from
GE Healthcare (Uppsala, Sweeden). Sequencing grade trypsin was obtained
from Promega Corporation (Madison, WI).
(DOPE: 18:1 (Δ9-Cis) was purchased from Avanti Polar Lipids
(Alabaster, AL). N-(3-(Dimethylamino)propyl)-N′-ethylcarbodiimide (EDC), N-hydroxysuccinimide
(NHS), 4-pentynoic acid, copper(II) sulfate pentahydrate,
were obtained from Sigma–Aldrich Chemical Co. (St. Louis, MO).
BCA Protein Assay Reagent and HPLC grade methanol and water were purchased
from Fisher Scientific (Pittsburgh, PA). Mouse mammary carcinoma (4T1)
cells were acquired from ATCC (Manassas, VA). Roswell Park Memorial
Institute (RPMI) medium 1640, 1× phosphate buffered saline (PBS),
fetal bovine serum (FBS), trypsin, and penicillin–streptomycin
were all purchased from Mediatech, Inc. (Manassas, VA). Plasmocin
was obtained from InvivoGen (San Diego, CA). Hoechst 33342 nucleic
acid stain, and DID (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine
perchlorate) was obtained from Life Technologies (Carlsbad, CA). Azide-Fluor
545 was purchased from KeraFAST, Inc. (Boston, MA). Formalin was purchased
from JT Baker (Center Valley, PA). Sepharose CL-4B was purchased from
GE Healthcare (Uppsala, Sweeden). Sequencing grade trypsin was obtained
from Promega Corporation (Madison, WI).
5
Molecular Biology Reagents and Protocols
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Designed oligonucleotides and DNA extraction kits (product codes: 27104 and 28104) were ordered from Qiagen. Expand High fidelity Taq polymerase (11732641001), T4 DNA ligase (10716359001), HindIII (10656321001), and BamHI (10 656 275 001) were from Roche Diagnostics (Mannheim, Germany). NdeI (R0111S) was from New England Biolabs and the dNTP mix (18427013) from Invitrogen. Tryptone (211921), yeast extract (212750), and sodium (2-sulfonatoethyl)-methanethiosulfonate (MTSES, AM3720 INTERCHIM) were from Thermo Fisher. Amberlite XAD-4 (06444), ampicillin (A9518), bathophenanthroline (146617), diamide (D3648), dithioerythritol (D8255), egg yolk phospholipids (lecithin from eggs, 61755), Na2SO4 (239313), N-dodecanoylsarcosine (sarkosyl, L512), N,N′-1,2-phenylenedimaleimide (o-PDM, 104590), N,N′1,3-phenylenedimaleimide (m-PDM, 160458), PIPES (P6757), phenylarsine oxide (PAO, P3075) pyridoxal-5’-phosphate (82870), sephadex G-75 (17-0050-01), and Tris base (10708976001) triton X-114 (93422) were from Sigma-Aldrich. The radioactive substrates 2-Oxo [1-14C]glutaric acid (NEC597) and 14C-NAD+ (NEC831) were purchased from Perkin Elmer, and [8-3H]GTP (TRK314) from Amersham Biosciences. All reagents were of analytical grade.
6
Ferritin Iron Mobilization Kinetics
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Ferritin (horse spleen) and NADPH-P 450 reductase (rabbit liver) were obtained from Sigma-Aldrich Co. (St. Louis, MO).
The standard reaction mixture for iron mobilization from Ferritin contains 1.9 μM Ferritin, 1U NADPH-P 450 reductase, 20 μM bathophenanthroline disulfonic acid 2Na salt in 20 mM phosphate buffer, pH 7.4. bathophenanthroline is used as a quantity regent of iron by binding to ferrous iron and forming orange-red chelate. We measured the quantity of released ferrous iron in real time using the spectrophotometer (U-3900, Hitachi High-Tsch Science Corporation, Japan) after adding 100 μM NADPH to the standard reaction mixture. The quantity of released ferrous iron was calculated from absorbance at 530 nm by using a molar absorbance coefficient of 22.1 × 10 3 M -1 cm -1 for bathophenanthroline.
To investigate the effect of superoxide dismutase on the reductive mobilization of iron, 10 μM superoxide dismutase added to the standard reaction mixture. In another experiment to elucidate the mechanism of Ferritin iron reduction, 2 mM ferricyanide or 6.7 μM cytochrome C was added each to the standard reaction mixture.
No less than 4 experiments were performed separately. The results are expressed as the representative data for all the experiments.
The standard reaction mixture for iron mobilization from Ferritin contains 1.9 μM Ferritin, 1U NADPH-P 450 reductase, 20 μM bathophenanthroline disulfonic acid 2Na salt in 20 mM phosphate buffer, pH 7.4. bathophenanthroline is used as a quantity regent of iron by binding to ferrous iron and forming orange-red chelate. We measured the quantity of released ferrous iron in real time using the spectrophotometer (U-3900, Hitachi High-Tsch Science Corporation, Japan) after adding 100 μM NADPH to the standard reaction mixture. The quantity of released ferrous iron was calculated from absorbance at 530 nm by using a molar absorbance coefficient of 22.1 × 10 3 M -1 cm -1 for bathophenanthroline.
To investigate the effect of superoxide dismutase on the reductive mobilization of iron, 10 μM superoxide dismutase added to the standard reaction mixture. In another experiment to elucidate the mechanism of Ferritin iron reduction, 2 mM ferricyanide or 6.7 μM cytochrome C was added each to the standard reaction mixture.
No less than 4 experiments were performed separately. The results are expressed as the representative data for all the experiments.
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