E. coli C43 (DE3) competent cells were independently transformed with the corresponding pET28 vectors, containing IsdH NEAT1-NEAT3, IsdH linker-NEAT3, IsdH NEAT3, or IsdB linker-NEAT2. Transformed cells were screened on Petri dishes with LB–agar containing kanamycin (50 μg ml−1; Wako). Expression of the heme-free proteins was carried out in M9 minimal medium supplemented with kanamycin (5 mg l−1) as described previously (16 (link)). To produce the heme-bound forms, transformed cells were grown in LB medium supplemented with 100 μM FeCl3 (Wako) and incubated at 37 °C (26 ). When the absorbance at 600 nm reached a value of 0.4, protein expression was induced by addition of 0.5 mM IPTG and cells were incubated for 20 h at 28 °C.
Fecl3
Manufactured by Fujifilm
Sourced in Japan
FeCl3 is a chemical compound consisting of one iron (Fe) atom and three chlorine (Cl) atoms. It is a solid, crystalline substance that is commonly used in various industrial and laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Zncl2, by Fujifilm (2 mentions)
Mgcl2, by Fujifilm (2 mentions)
Kanamycin, by Fujifilm (1 mentions)
Alcl3, by Fujifilm (1 mentions)
Penicillin, by Nacalai Tesque (1 mentions)
Streptomycin, by Nacalai Tesque (1 mentions)
Cocl2, by Fujifilm (1 mentions)
Cuso4, by Fujifilm (1 mentions)
Nicl2, by Fujifilm (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Methanol, by Fujifilm (1 mentions)
Fecl3, by Merck Group (1 mentions)
Sodium pyruvate, by Fujifilm (1 mentions)
Human serum albumin (hsa), by Fujifilm (1 mentions)
N n diethyl p phenylenediamine, by Fujifilm (1 mentions)
Bolt reducing agent, by Thermo Fisher Scientific (1 mentions)
Precision plus protein dual color standard, by Bio-Rad (1 mentions)
Ohpak sb 804 hq column, by Resonac (1 mentions)
Bolt mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
9 protocols using fecl3
1
Recombinant Expression of Heme-Binding Proteins
2
Metal Exposure Effects on HepG2 Cells
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The cell lines present in this study were obtained from RIKEN BRC CELL BANK. HepG2 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS, Biowest), 100 U mL−1 penicillin, and 100 U mL−1 streptomycin (Nacalai Tesque) at 37°C in a 5% CO2 atmosphere. The metal exposure was performed as follows: 2 × 104 cells were seeded in a 3.5-cm culture dish and first cultured with normal medium for 48 h; subsequently, they were washed with phosphate-buffered saline (PBS) and then treated with medium including FeCl3, AlCl3, CoCl2, CuSO4, NiCl2 and ZnCl2 (FUJIFILM Wako Pure Chemical Corporation), ranging from 20 to 200 μM, separately. After incubation for 24 h, the cells were fixed using the following steps. First, cells were digested with .25% trypsin/EDTA, followed by PBS washing. Then, the cells were gently suspended in a hypotonic solution (75 mM KCl) and allowed to stand for 8 min at room temperature. After the same volume of Carnoy’s solution (fixative solution, methanol/acetic acid (3/1, v/v)) was added, cells were mixed gently. After centrifugation at 1,500 rpm, the supernatant was discarded, and an equal volume of fresh Carnoy’s solution was added. This procedure was repeated twice. Fixed cells were stored in Carnoy’s solution at −20°C.
3
Copper Etching via FeCl3 Solution
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4
Assessing P. aeruginosa EVs Inhibition on S. aureus
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Growth inhibition assay in P. aeruginosa culture supernatant and TSB supplemented with PaEVs was performed. To prepare P. aeruginosa supernatant, P. aeruginosa was cultured in TSB for 8 h, and the bacterial cells were removed from the culture supernatant by centrifugation and filtration as described above. The absence of bacterial cell contamination was confirmed by inoculating a portion of the supernatant into TSB and TSA and incubating at 37°C for 48 h. S. aureus as well as other bacterial and yeast cells were inoculated into P. aeruginosa culture supernatant or TSB containing 0–5 μg/mL purified PaEVs by adjusting to 5 × 103 CFU/mL and incubated at 37°C for 0–12 h. Growth of each microorganism was monitored by measuring OD at 600 nm and/or the microbial numbers were enumerated by plate count assay on TSA. The growth of PaEV-treated S. aureus in the presence of 50 and 100 μM FeCl3 or 15 mM sodium pyruvate (FUJIFILM Wako Pure Chemical Industries, Osaka, Japan) supplementation was also assessed by the same method as described above. For the effect of PaEVs under oxygen supplementation, S. aureus was cultured in the presence of 1 μg/mL PaEVs at 125 rpm shaking.
5
Investigating HDL Levels with ELISA Assay
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Fe(NH4)2(SO4)2, FeCl3, N,N′-diethyl-p-phenylenediamine, dextran sulfate sodium salt (molecular weight range, 36,000–50,000), MgCl2, and human serum albumin were purchased from Wako (Tokyo, Japan). Tert-butyl hydroperoxide (70% in water) was purchased from TCI (Tokyo, Japan). NaCl was from Sigma-Aldrich (Tokyo, Japan). HDL Human ELISA (enzyme-linked immunosorbent assay) Kit (ab125961) came from Abcam (Cambridge, UK). A Bolt LDS sample Buffer, Bolt Reducing Agent, and Bolt MOPS SDS Running Buffer were purchased from the Life Technologies Corporation (Carlsbad, CA, USA). The Precision Plus Protein Dual Color Standards were from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The OH pak SB-804HQ column was obtained from Showa Denko KK (Tokyo, Japan).
6
Tyrosinase-Mediated Antioxidant Evaluation
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Tyrosinase (from mushrooms, specific activity 1715 U/mg), Ampliflu™ Red reagent (1-acetyl-3,7-dihydroxyphenoxazine), DA, BSA, βLG, MeCA, l -Cys and NAC were purchased from Sigma-Aldrich (St. Louis, MO, USA). FeCl3, phenol, 3,5-di-tert-butyl-1,2-benzoquinone (DBBQ), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), 2-mercaptoethanol (thioglycol), reduced glutathione (GSH), thioglycolic acid, n-hexane, H2O2, EDTA·2Na and methanol (HPLC grade) were purchased from Wako Pure Chemical Industry (Osaka, Japan). DPRA(Cys) (Ac-RFAACAA-COOH) and DPRA(Lys) (Ac-RFAAKAA-COOH) were purchased from Scrum Inc. (Tokyo, Japan). CuSO4·5H2O and HClO4 were from Katayama Chemical Co. Ltd. (Osaka, Japan). NACHis, NACLeu, NACLys and dimethyl sulfoxide (DMSO) were from Tokyo Chemical Industry Co. (Tokyo, Japan). All other chemicals were of the highest purity commercially available. The highest purity Milli-Q water (Milli-Q Advantage, Merck Millipore Co., Tokyo, Japan) was used in order to avoid contamination of metal ions. The number of thiol group in BSA and βLG were determined to be 0.30 and 0.59 mol/mol protein, respectively, using the method of Elleman [51 (link)] with DTNB. The number of thiol group in BSA was close to 0.38 mol/mol BSA [30 (link)].
7
Immobilized Metal Affinity Chromatography
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IMAC analysis was done according to a previous study [38 (link)]. Vanadyl sulfate (VOSO4·nH2O, n = 3–4; 99.9%), along with MgCl2, CaCl2, MnCl2, FeCl3, CoSO4, CuCl2, and ZnCl2, were purchased from Fujifilm Wako Pure Chemical Corporation, Japan. Each metal was dissolved in deionized water (DW) at 1 mM and stored at room temperature.
Chelating Sepharose FF resin (250 μL; Thermo Fisher Scientific, USA) was washed with water and mixed with each metal solution in a 2 mL plastic tube. The resin was washed twice with distilled water and twice with binding buffer (100 mM NaCl, 20 mM Na phosphate, pH 7.4). Protein in binding buffer (∼100 μg mL−1) was mixed with the resin by rotation for 30 min at room temperature. Non-bound proteins were removed by centrifugation, then the resin was washed twice with binding buffer. Bound proteins and metal ions were eluted with 50 mM EDTA (pH 8.0). Non-bound and bound protein fractions were analyzed by SDS-PAGE and CBB staining.
Chelating Sepharose FF resin (250 μL; Thermo Fisher Scientific, USA) was washed with water and mixed with each metal solution in a 2 mL plastic tube. The resin was washed twice with distilled water and twice with binding buffer (100 mM NaCl, 20 mM Na phosphate, pH 7.4). Protein in binding buffer (∼100 μg mL−1) was mixed with the resin by rotation for 30 min at room temperature. Non-bound proteins were removed by centrifugation, then the resin was washed twice with binding buffer. Bound proteins and metal ions were eluted with 50 mM EDTA (pH 8.0). Non-bound and bound protein fractions were analyzed by SDS-PAGE and CBB staining.
8
Heme-Bound IsdH Protein Expression in E. coli
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Escherichia coli C43 (DE3) competent cells were separately transformed with the expression vectors of the aforementioned IsdH constructs. Expression of heme-depleted protein was carried out in M9 minimal medium supplemented with kanamycin (5 mg L−1) as previously described (14 (link)). To produce the heme-bound form of IsdH (except for the thermostability assays, and the heme transfer experiments between NEAT domains), E. coli C43 (DE3) cells were transformed with the corresponding vector. Bacterial growth was carried out in LB medium supplemented with 100 μM FeCl3 (Wako) at 37 °C. When the absorbance at 600 nm reached a value of 0.4, expression of IsdH was induced by the addition of 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and kept for 20 h at 28 °C under vigorous shaking.
9
Synthesis and Characterization of Graphene Oxide-Metal Composites
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Graphene oxide powder was purchased from Nanjing ICNANO Technology Company (Nanjing, China); HAuCl4•4H2O and 2-propanol were purchased from Wako Pure Chemical Industries (Osaka, Japan). The atomic absorption standard solutions (1000 μg/mL) of Cr(VI), Cr(III), Cu(II), Pb(II), Cd(II), Zn(II), Co(II) and Mn(II) were purchased from Nacalai Tesque (Kyoto, Japan). The standard stock solution of Fe(III) was prepared by dissolving an adequate amount of FeCl3 (Wako, Osaka, Japan) in distilled water. Perchloric acid (HClO4) was obtained from Nacalai Tesque (Kyoto, Japan). All chemicals were of analytical grade, and solutions were prepared with distilled water purified by a WS200 distillation system (Yamato Scientific Co., Tokyo, Japan).
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