8453 diode array spectrophotometer

Manufactured by Agilent Technologies

Sourced in United States, Italy, Japan

The 8453 diode array spectrophotometer is a lab equipment product manufactured by Agilent Technologies. It is designed to measure the absorbance of light by samples over a wide range of wavelengths. The core function of this instrument is to provide accurate and reliable spectral data for various applications.

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72 protocols using 8453 diode array spectrophotometer

Peptide Purification and Characterization

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UV-Vis spectra were recorded on an Agilent (Santa Clara, CA, USA) 8453 diode array spectrophotometer equipped with a magnetically stirred quartz optical cell of 1 cm path length. Peptide purification was performed on a Shimadzu HPLC instrument equipped with two LC-20AD pumps and an SPDM20A diode array detector (working range: 190–800 nm) using a Phenomenex Jupiter 4U Proteo semipreparative column (4 μm, 250 × 10 mm). Mass spectrometry analysis was performed on an LCQ ADV MAX ion-trap mass spectrometer with an ESI ion source. The ESI conditions were as follows: capillary temperature 210 °C, tube lens voltage −25 V, and source voltage +4.9 kV. The system was run in automated LC-MS/MS mode, using a surveyor HPLC system (Thermo Finnigan, San Jose, CA, USA) equipped with a Phenomenex Jupiter 4U Proteo column (4 μm, 150 × 2.0 mm). For the analysis of peptide fragments, Bioworks 3.1 and Xcalibur 2.0.7 SP1 software were used (Thermo Finnigan, San Jose, CA, USA).

De Caro S., De Soricellis G., Dell’Acqua S., Monzani E, & Nicolis S. (2023). Biological Oxidations and Nitrations Promoted by the Hemin–Aβ16 Complex. Antioxidants, 12(7), 1319.

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Absorbance Spectrum of LaFeCoO3 Thin Film

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An absorbance spectrum of the
LaFe_0.4Co_0.6O₃ thin film was measured
with an Agilent 8453 diode-array spectrophotometer at several applied
potentials using the bare substrate as a blank. The film-coated FTO
working electrode, platinum mesh counter electrode, and SCE reference
electrodes were placed in a flat-sided cell filled with 0.1 M Na₂SO₄. The cell was the bottom three-fourths of a
Corning polystyrene tissue culture flask with the top machined off.
The applied potential was controlled using a Pine Instruments bipotentiostat.

Dervishogullari D., Sharpe C.A, & Sharpe L.R. (2017). LaFexCo(1–x)O3 Thin-Film Oxygen Reduction Catalysts Prepared Using Spray Pyrolysis without Conductive Additives. ACS Omega, 2(11), 7695-7701.

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Isopentenyl Diphosphate Isomerase-2 Purification

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apo-sp-IDI-2 was incubated with 2 mM FMN, overnight at 4 °C. Reconstituted holoenzyme was loaded onto a Ni-IDA affinity chromatography column (PrepEase, Affymetrix) and washed with lysis-equilibrium-wash buffer (50 mM NaH₂PO₄, pH 8.0, 300 mM NaCl) until the wash no longer appeared yellow (three washes). Protein was eluted with 50 mM NaH₂PO₄, pH 8.0, 300 mM NaCl, 250 mM imidazole and analyzed by UV-vis spectroscopy (Agilent 8453 diode array spectrophotometer) and SDS-PAGE. Fractions containing IDI-2 were concentrated and combined.
The extinction coefficient for the flavoprotein was determined by the procedure of Macheroux.^{[22 ]} Briefly, the absorption of a 75 μM solution of sp-IDI-2 in 10 mM Tris, pH 8.0 was measured at 445 nm. To this sample, 50% trichloroacetic acid (final concentration, ~8%) was added, upon which the protein precipitated. After centrifugation, the pH was adjusted to 8.0 using solid Na₂CO₃. The absorbance at 450 nm (ε_FMN = 12,200 M⁻¹cm⁻¹, ^{[23 ]}) of the supernatant was measured and used to determine the extinction coefficient of FMN bound sp-IDI-2.

de Ruyck J., Janczak M.W., Neti S.S., Rothman S.C., Schubert H.L., Cornish R.M., Matagne A., Wouters J, & Poulter C.D. (2014). Determination of kinetics and crystal structure of a novel Type 2 Isopentenyl Diphosphate: Dimethylallyl Diphosphate Isomerase from Streptococcus pneumoniae. Chembiochem : a European journal of chemical biology, 15(10), 1452-1458.

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Characterization of Tau Peptide Modifications

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All reagents for the peptide synthesis, including protected amino acids and rink amide resin, were purchased from Novabiochem, while other chemical compounds were reagent grade from Sigma-Aldrich. Purification of tau peptides was performed on a Shimadzu HPLC instrument equipped with two LC-20AD pumps and a SPD-M20A diode array detector, using a Phenomenex Jupiter 4µ Proteo semipreparative column (4 μm, 250 × 10 mm). Mass analysis and LC-MS/MS chromatograms were acquired with a LCQ ADV MAX ion-trap mass spectrometer, with an ESI ion source. The instrument works in automated LC-MS/MS mode through a surveyor HPLC system (Thermo Finnigan, San Jose, CA, USA) equipped with a Phenomenex Jupiter 4µ Proteo column (4 µm, 150 × 2.0 mm). In order to identify the oxidative modifications of peptide fragments, Bioworks 3.1 and Xcalibur 2.0.7 SP1 software were used (Thermo Finnigan, San Jose, CA, USA). UV-visible absorption spectra were collected with a Thermo Evolution 260 Bio spectrophotometer, provided with a Peltier thermostat. Circular dichroism spectra were acquired with a Jasco J715 spectropolarimeter, equipped with a Peltier thermostat. UV-visible kinetic profiles were obtained through an Agilent 8453 diode array spectrophotometer, equipped with a thermostated, magnetically stirred optical cell.

Bacchella C., Gentili S., Mozzi S.I., Monzani E., Casella L., Tegoni M, & Dell’Acqua S. (2022). Role of the Cysteine in R3 Tau Peptide in Copper Binding and Reactivity. International Journal of Molecular Sciences, 23(18), 10726.

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Spectroscopic Analysis of Anthocyanin-Copigment Interactions

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Spectra were recorded with an Agilent 8453 diode array spectrophotometer fitted with a quartz cell (optical path length = 1 cm) equipped with a stirring magnet. A constant temperature of 25 °C in the cell was obtained by use of a water-thermostated bath.
Small volumes of concentrated PA solutions in acidified MeOH (0.1 M HCl, 100% flavylium) were added to a 0.2 M acetate buffer containing 12% EtOH or to a citrate-phosphate buffer (pH range 1–6). In the cell, the pigment concentration was ca. 50 µM for PA1 and 25 or 10 µM for PA2. The spectral monitoring was carried out over 15–30 min. Then, the samples were collected and kept overnight at room temperature to repeat the spectroscopic analysis. The experiments were also repeated with buffers containing a copigment (CP: chlorogenic acid or catechin) in large excess (20–100 equiv.) or a metal ion (PA1 + 10 equiv. AlCl₃ or Fe(NO₃)₃) in acetate buffer + 12% EtOH.
Alternatively, some samples without CP were combined at the end of the kinetic monitoring and the pH was adjusted to 4.5 or 5.2. To the resulting solutions, the copigment was added (CP-to-PA molar ratios = 20–100) and the UV-visible spectra were recorded.

Vallverdú-Queralt A., Biler M., Meudec E., Guernevé C.L., Vernhet A., Mazauric J.P., Legras J.L., Loonis M., Trouillas P., Cheynier V, & Dangles O. (2016). p-Hydroxyphenyl-pyranoanthocyanins: An Experimental and Theoretical Investigation of Their Acid—Base Properties and Molecular Interactions. International Journal of Molecular Sciences, 17(11), 1842.

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Multimodal Analytical Techniques in Cellular Research

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UV-visible spectroscopy was performed with an Agilent Hewlett-Packard 8453 diode-array spectrophotometer equipped with an Agilent 89090A thermostat. Absorbance and fluorescence measurements of 96 well plates were accomplished with a Perkin-Elmer HTS 7000 Bio Assay reader. Cells were counted using a Beckman Coulter Z-2 cell and particle counter. Cell viability and angiogenesis was monitored using a Nikon eclipse TS-100 inverted microscope. An Eppendorf thermocycler was used to prepare cDNA. Sequence detection was then carried out using 7300 Real-Time PCR System from Applied Biosystems. An iBlot^® gel transfer device from Invitrogen, Carlsbad, CA was used for dry transfer in Western blots. DNA damage was assessed using a Comet Assay^® Electrophoresis System (Trevigen Inc. Gaithersburg, MD).

Basudhar D., Cheng R.C., Bharadwaj G., Ridnour L.A., Wink D.A, & Miranda K.M. (2015). Chemotherapeutic Potential of Diazeniumdiolate-based Aspirin Prodrugs in Breast Cancer. Free radical biology & medicine, 83, 101-114.

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Optical and Spectroscopic Characterization

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UV–VIS analysis was carried out using an Agilent 8453 diode array spectrophotometer [44 (link)]. Fluorescence study was carried out using a Jasco FP-750 spectrofluorometer (JASCO, Tokio, Japan). Quantum yield was determined by the comparative method described in our previous article [44 (link)]. The study PL-pH dependence was carried out at pH = 4, 5, 6, 7, 8, 9. The pH was adjusted using an Elmetron CX-551 pH-meter (combined glass pH electrode, Elmetron, Zabrze, Poland). The solvatochromism study was carried out using the following solvents: water, acetone, acetonitrile, tetrahydrofuran, and methanol. Infrared analysis was performed using a Nexus 470 diamond crystal ATR FTIR spectrophotometer (Thermo Fisher Scientific (Waltham, MA, USA).

Janus Ł., Radwan-Pragłowska J., Piątkowski M, & Bogdał D. (2020). Smart, Tunable CQDs with Antioxidant Properties for Biomedical Applications—Ecofriendly Synthesis and Characterization. Molecules, 25(3), 736.

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Spectroscopic Characterization of Ruthenium Porphyrin Complexes

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Acetonitrile and methylene chloride were obtained from Fisher Scientific and distilled over P₂O₅ prior to use. All organic substrates for LFP kinetic studies were the best available purity from Aldrich Chemical Co. and were passed through a dry column of active alumina (Grade I) before use. Pyridine N-oxide was obtained from Aldrich and used as such. 2,6-Dichloropyridine N-oxide was prepared by oxidation of the corresponding pyridine precursors by H₂O₂ (50%) in trifluoroacetic acid according to the known procedure.⁵⁰ 5,10,15,20-Tetraphenylporphyrin free ligand (H₂TPP)⁵¹ and its ruthenium(II) carbonyl complex Ru^II(TPP)(CO) (6) were prepared by literature methods.^{48 (link)} Complexes 2 were prepared according to the literature procedure⁴² and purified by chromatography on basic alumina. All the compounds were characterized by UV-vis, ¹H NMR and IR spectra, matching those reported data.
UV-vis spectra were recorded on an Agilent 8453 diode array spectrophotometer. IR spectra were obtained on a Bio-Rad FT-IR spectrometer. NMR was performed on a JEOL ECA-500 MHz spectrometer at 298K with tetramethylsilane (TMS) as internal standard. Chemical shrifts (ppm) are reported relative to TMS. X-band ESR spectra were recorded on a Varian E109E spectrometer equipped with a low-temperature dewar.

Zhang R., Vanover E., Luo W, & Newcomb M. (2014). Photochemical generation and kinetic studies of a putative porphyrin-ruthenium(V)-oxo species. Dalton transactions (Cambridge, England : 2003), 43(23), 8749-8756.

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Quantitative NQO1 Activity Assay

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NQO1 activity was measured in 50 mM HEPES-KOH, pH 7.4. A reaction mixture containing recombinant NQO1 and 0.5 mM NADH was incubated at 30 °C for 5 min in a 1 cm path length quartz cuvettes in a thermostatized Agilent 8453 diode-array spectrophotometer. The reaction was triggered by the addition of DCPIP 75 μM as the electron acceptor. Initial reaction rates were determined from changes in A_600nm resulting from the reduction of DCPIP and corrected for the non-enzymatic reaction. The NQO1 concentration used varied depending on the variant to ensure linearity over time and protein concentration: 1–2 nM for WT and 25–50 nM for p.P187S. The specific activity was calculated using ε₆₀₀ _nm = 21000 M⁻¹·cm⁻¹ for DCPIP.

Medina-Carmona E., Palomino-Morales R.J., Fuchs J.E., Padín-Gonzalez E., Mesa-Torres N., Salido E., Timson D.J, & Pey A.L. (2016). Conformational dynamics is key to understanding loss-of-function of NQO1 cancer-associated polymorphisms and its correction by pharmacological ligands. Scientific Reports, 6, 20331.

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Production and Characterization of 2-HMS

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ACMS was generated by catalysing the insertion of molecular oxygen to 3-hydroxyanthranilic acid by purified, Fe²⁺ reconstituted 3-hydroxyanthranilate 3,4-dioxygenase as described previously16 (link)20 (link). 2-HMS is generated non-enzymatically from ACMS following a previously established method24 (link). The pH of solutions containing ACMS was adjusted to ~2 by the addition of hydrochloric acid. 2-HMS formation was monitored on an Agilent 8453 diode-array spectrophotometer at 315 nm. The solutions were then neutralized with sodium hydroxide once the absorbance at 315 nm stopped increasing. 2-HMS at neutral pH has a maximum absorbance at 375 nm (ref. 24 (link)).

Huo L., Davis I., Liu F., Andi B., Esaki S., Iwaki H., Hasegawa Y., Orville A.M, & Liu A. (2015). Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action. Nature Communications, 6, 5935.

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