Quantamaster spectrofluorometer

Manufactured by Horiba

Sourced in Japan, United Kingdom, United States

The QuantaMaster Spectrofluorometer is a high-performance fluorescence spectrometer designed for accurate and sensitive measurements of fluorescence properties in a variety of samples. It provides the core function of measuring the emission spectra and fluorescence intensity of samples, enabling researchers to analyze their fluorescent characteristics.

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Lab products found in correlation

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Close spelling variants of this product

Spectrofluorometer (35 citations) QuantaMaster (20 citations) QuantaMaster QM-4 spectrofluorometer (12 citations) QuantaMaster 40 spectrofluorometer (9 citations) QuantaMaster C-60/2000 spectrofluorometer (3 citations) PTI QuantaMaster 400 spectrofluorometer (2 citations) PTI QuantaMaster spectrofluorometer (2 citations)

26 protocols using quantamaster spectrofluorometer

Fluorescence Spectroscopy of Membrane Transporters

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The ability of DDM and
A8-35 solubilized Mhp1 and GalP to bind known targets was assessed
by fluorescence emission spectroscopy on a QuantaMaster spectrofluorometer
(Photon Technology International, Ford, West Sussex, UK), using previously
published methods.^{47 (link)} Briefly, purified
Mhp1 or GalP (200 μg/mL) solubilized in either 0.02% (w/v) DDM
or by addition of a 1:5 (w/w) excess of A8-35 in 100 mM NH₄HCO₃, pH 8.0, were analyzed at 20 °C. Tryptophan
fluorescence of protein samples was excited at 295 nm, and the intrinsic
fluorescence emission at 330 nm was monitored. Micromolar additions
of ligand (l-benzylhydantoin for Mhp1 and forskolin for GalP)
were performed from 0 to 2 mM (Mhp1) or 0–100 μM (GalP).
Samples were mixed for 1 min after each addition before measuring
the fluorescence emission spectrum. Nonlinear regression analysis
was performed using GraphPad Prism 6 (Graphpad Software, San Diego,
CA, USA).

Calabrese A.N., Watkinson T.G., Henderson P.J., Radford S.E, & Ashcroft A.E. (2014). Amphipols Outperform Dodecylmaltoside Micelles in Stabilizing Membrane Protein Structure in the Gas Phase. Analytical Chemistry, 87(2), 1118-1126.

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2-Aminopurine Fluorescence Measurements

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All oligonucleotides used in these experiments were first ethanol-precipitated and resuspended to remove impurities from commercial synthesis that interfered with the optical spectra of interest. Oligonucleotides were combined to their final concentration in 1X nucleic acid spectroscopy buffer (10 mM K₂HPO₄/KH₂PO₄, pH 6.7, 150 mM KCl, 0.1 mM EDTA) and annealed on a thermocycler (95°C to 25°C over the course of 40 min). Final concentrations were 5 µM 2-AP-containing oligonucleotide, 5.5 µM complementary oligonucleotide (if present), 6 µM same-stranded oligonucleotide (if present), and 6 µM RNA oligonucleotide (if present). Samples were placed in a 1.5-mm fluorescence cuvette (Hellma Analytics) and allowed to equilibrate inside the temperature-controlled (30°C) cell of a QuantaMaster spectrofluorometer (Photon Technology International) for 3 min. The lamp power was set to 74 W, and the slit widths were set as follows (excitation slit 1: 0.5 mm; excitation slit 2: 1 mm; emission slit 1: 2 mm; emission slit 2: 0.9 mm). Fluorescence intensity (λ_ex=310 nm (4 nm bandpass), λ_em=370 nm (3.6 nm bandpass)) was measured for 30 s, and the average across those 30 s was reported. Oligonucleotide identities and sequences are shown in Supplementary file 1.

Cofsky J.C., Karandur D., Huang C.J., Witte I.P., Kuriyan J, & Doudna J.A. (2020). CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks. eLife, 9, e55143.

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Protein Unfolding Thermodynamics by Fluorescence

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Guanidinium chloride (GdmCl) was used as the denaturant. Protein solutions at varying GdmCl concentrations were prepared and equilibrated for one hour at room temperature before measuring changes in fluorescence signals as a function of the denaturant concentration. Concentration of the denaturant was determined using refractive index measurements^{30 (link)}. For measuring changes in the 280 nm (aromatic) fluorescence, 3 µM IFNA2 in formulation buffer was used with varying concentrations of BA. Fluorescence spectra were obtained using a Quantamaster spectrofluorometer (Photon Technology International, Birmingham, New Jersey) with 4 nm slits. The ΔG values were determined by fitting the changes in optical signals at different denaturant concentrations to a two-state unfolding model^{31 (link)},^{32 (link)}. The m-value, which is the slope of linear variation of ΔG with denaturant concentration, was obtained by fitting the data to a Santero-Bolen two-state equation^{31 (link)},^{32 (link)}.

Bis R.L., Singh S.M., Cabello-Villegas J, & Mallela K.M. (2014). ROLE OF BENZYL ALCOHOL IN THE UNFOLDING AND AGGREGATION OF INTERFERON α-2A. Journal of pharmaceutical sciences, 104(2), 407-415.

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Biotin-Fluorescent Probe Dissociation Kinetics

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The dissociation rate constant (k_diss) of fluorescently labelled biotin was determined by fluorescence spectrometry using the biotin-labelled fluorescent probe ArcDia BF560 (ArcDia, Turku, Finland) as described in [34] (link). In principle, the changes in the fluorescence intensity of 50 nM dye in 50 mM sodium phosphate, 650 mM NaCl, 0.1 mg/ml BSA, pH 7 was measured after the addition of biotin-binding protein to a final monomer concentration of 100 nM. The dissociation of this complex was observed by addition of a 100-fold molar excess of free biotin. The assay was performed at 25°C using a QuantaMaster Spectrofluorometer (Photon Technology International, Inc., Lawrenceville, NJ, USA) equipped with circulating water bath thermostat. The fluorescence probe was excited at 560 nm and emission was measured at 578 nm.

Taskinen B., Airenne T.T., Jänis J., Rahikainen R., Johnson M.S., Kulomaa M.S, & Hytönen V.P. (2014). A Novel Chimeric Avidin with Increased Thermal Stability Using DNA Shuffling. PLoS ONE, 9(3), e92058.

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Oligomerization Dynamics of ms-SpoIIEcyt

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To study the oligomerization of ms-SpoIIE_cyt in the presence and absence of Mn²⁺, the fluorescence spectra of ms-SpoIIEcyt-Cy5 were acquired in the presence of Mn²⁺ or EDTA. ms-SpoIIE_cyt was purified from lysates by binding to an amylose resin as described above, with the inclusion of an on-column Cy5 labeling step. After washing with buffer A, the resin with bound ms-SpoIIE_cyt was mixed with fluorescent label Cy5 (GE Healthcare) and incubated for 1 hour at 4°C. Excess label was washed away using the same buffer and labeled protein was eluted with buffer A supplemented with 10 mM maltose.
Equal volumes of ms-SpoIIE_cyt-Cy5 were titrated to a buffer containing 50 mM Tris/HCl, 300 mM KCl, 1 mM EDTA with or without 10 mM MnCl₂, at 30°C. Each titration step increased the total protein concentration with 0.23 μM. Immediately after each addition of ms-SpoIIE_cyt, a fluorescence spectrum (640–685 nm, excitation 633 nm) was recorded in a QuantaMaster^™ spectrofluorometer controlled by the FelixGX program (Photon Technology International, Inc.). Oligomerization was monitored by Cy5 self-quenching and the titration was stopped when fluorescence no longer increased upon the addition of protein.

Cendrowicz E., de Sousa Borges A., Kopacz M, & Scheffers D.J. (2017). Metal-dependent SpoIIE oligomerization stabilizes FtsZ during asymmetric division in Bacillus subtilis. PLoS ONE, 12(3), e0174713.

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Quantifying Unconjugated Small Molecule Binding Affinity

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The binding affinity of the unconjugated small molecules to antidins was determined utilizing the intrinsic fluorescence originating from the aromatic amino acid residues (mainly tryptophan and tyrosine) of Avd and the fluorescence quenching caused by ligand binding. In brief, 100 nM protein samples in 50 mM NaH₂PO₄/Na₂HPO₄, 650 mM NaCl, pH 7 were excited at 280 nm, and emission was collected at 350 nm using QuantaMaster Spectrofluorometer (Photon Technology International, Inc.) with 2 nm slits. The assay was performed in a quartz cuvette with stirring at 25°C. The ligand was added to the protein sample in small aliquots (6–50 000 nM) and the fluorescence intensity was monitored after a short incubation. The dissociation constant (K_d) was determined from the resulting quenching curve using GraphPad Prism (GraphPad Software, Inc.). The data were fitted to a quadratic equation [72 ] for tight binding interactions, which takes ligand depletion and nonspecific binding into account (described in detail in [38 (link)]) (Fig 6).

Agrawal N., Lehtonen S.I., Uusi-Mäkelä M., Jain P., Viitala S., Määttä J.A., Kähkönen N., Azizi L., Riihimäki T.A., Kulomaa M.S., Johnson M.S., Hytönen V.P, & Airenne T.T. (2019). Molecular features of steroid-binding antidins and their use for assaying serum progesterone. PLoS ONE, 14(2), e0212339.

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Steady-State Fluorescence Emission Measurements

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All steady-state florescence emission measurements were performed at 37°C using a QuantaMaster spectrofluorometer (Photon Technology International, West Sussex, UK). Excitation and emission bandwidths were set at 4–8 nm. Tryptophan fluorescence was excited at 290 nm, and fluorescence emission was monitored from 300–500 nm, while the excitation wavelength for carboxyfluorescein (CF) was set to 492 nm and emission was monitored from 500–625 nm or 513 nm for continuous kinetic experiments.

Goodchild S.C., Sheynis T., Thompson R., Tipping K.W., Xue W.F., Ranson N.A., Beales P.A., Hewitt E.W, & Radford S.E. (2014). β2-Microglobulin Amyloid Fibril-Induced Membrane Disruption Is Enhanced by Endosomal Lipids and Acidic pH. PLoS ONE, 9(8), e104492.

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Membrane expansion visualization by R18 liposomes

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To visualize
membrane expansion, Octadecyl Rhodamine B chloride (R18) (Biotium
Inc. Fremont, USA) was incorporated into the liposomes at a concentration
of 5 mol % during liposome preparation. Synthesis reactions were performed
in buffer A with addition of 0.5 μM FadD, 50 μM CoA, 2700
μM oleic acid, 2.67 mM R18 liposomes (DOPC, DOPG, DOPE), 10
mM G3P, 0.5 μM PlsB, 1.5 μM PlsC and, if present 4 mM
ATP and 1% Triton X-100. Reactions were quenched with 10 mM EDTA,
and fluorescence was excited at 540 nm and emission spectra (560–680
nm) recorded using a QuantaMaster spectrofluorometer controlled by
the FelixGX program (Photon Technology International, Inc.).

Exterkate M., Caforio A., Stuart M.C, & Driessen A.J. (2017). Growing Membranes In Vitro by Continuous Phospholipid Biosynthesis from Free Fatty Acids. ACS Synthetic Biology, 7(1), 153-165.

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Mitochondrial Calcium Retention Capacity

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Calcium retention was evaluated fluorometrically using a QuantaMaster Spectrofluorometer (QM-400, Horiba Scientific, Kyoto, Japan) in a 200µL reaction volume at 37°C as previously described (34 (link)), with some modification. Assay buffer was Buffer E (0.25M sucrose, 10mM Trizma-HCl, 20mM Trizma-base, 10mM KH₂PO₄, 0.5mg/mL bovine serum albumin, 5mM Cr, 40µM EGTA, 1U/mL HK, 5mM glucose; pH=7.1) supplemented with 1µM calcium green 5N to fluorescently measure extracellular calcium (Ex/Em: 506/532). Mitochondria (50µg) were energized with S/Rot and 50µM ADP, then calcium was titrated in 30µM additions until mitochondrial permeability transition pore (MPTP) opening (rapid increase in fluorescence trace). Additional 100µM and 30µM additions were made following MPTP opening to confirm no additional calcium uptake. The assay was then ended with the addition of 10mM EGTA. Calcium retention capacity was considered the amount of calcium added prior to MPTP opening.

McLaughlin K.L., Nelson M.A., Coalson H.S., Hagen J.T., Montgomery M.M., Wooten A.R., Zeczycki T.N., Vohra N.A, & Fisher-Wellman K.H. (2022). Bioenergetic Phenotyping of DEN-Induced Hepatocellular Carcinoma Reveals a Link Between Adenylate Kinase Isoform Expression and Reduced Complex I-Supported Respiration. Frontiers in Oncology, 12, 919880.

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Simultaneous Determination of NAD+/NADH and ΔΨ

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NAD⁺/NADH and ΔΨ were determined simultaneously using a QuantaMaster Spectrofluorometer (QM-400, Horiba Scientific, Kyoto, Japan) as previously described (25 (link)), with some modifications. Experiments were performed with tissue mitochondria (20µg) in a 200µL reaction volume at 37°C. Assay buffer was Buffer D supplemented with Cr (5mM) and tetramethyl rhodamine methyl ester (TMRM; 0.2 µM). Mitochondria were stimulated using the creatine kinase clamp and ΔG_ATP was titrated via PCr additions (6, 15, 21mM). Oligomycin (0.02µM) was added to inhibit ATP synthesis, and cyanide (CN, 10mM) was added to induce 100% reduction of the matrix NADH pool, followed by isocitrate (5mM) to induce 100% reduction of the matrix NADPH pool. NAD⁺/NADH was detected at Ex/Em: 350/450 and expressed as a percentage reduction of the CN value as previously described (25 (link)). TMRM-derived ΔΨ was quantified by taking the fluorescence ratio of Ex/Em 576/590 to 551/590 and converting this to mV values using a tissue-specific standard curve determined as previously described (23 (link)).

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