Sx20 stopped flow

Manufactured by Applied Photophysics

The SX20 Stopped-Flow is a high-performance instrument designed for rapid mixing and analysis of biochemical and chemical reactions. It provides a precise and controlled environment for measuring kinetic processes with millisecond time resolution. The instrument is capable of automatically mixing small volumes of solutions and recording the resulting changes in optical properties over time.

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Lab products found in correlation

Akta prime purification system, by GE Healthcare (5 mentions) Affinity column his trap ff, by GE Healthcare (4 mentions) Sds page apparatus, by Bio-Rad (2 mentions) Nahco3, by Merck Group (2 mentions) Western blot apparatus, by Bio-Rad (2 mentions) Histrap ff, by GE Healthcare (1 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions) Pd 10 desalting column, by GE Healthcare (1 mentions) Spectramax 340pc plate reader, by Molecular Devices (1 mentions) Phosphate buffer, by Merck Group (1 mentions) Epoch plate reader, by Agilent Technologies (1 mentions) Sds page, by Bio-Rad (1 mentions)

Spelling variants of this product

SX20 stopped-flow spectrometer (42 citations) SX20 stopped-flow spectrophotometer (32 citations) SX20 stopped-flow instrument (14 citations) SX20 stopped-flow fluorimeter (5 citations) SX20 stopped-flow system (4 citations) SX20 stopped-flow UV-vis spectrometer (2 citations)

13 protocols using sx20 stopped flow

Protein Purification Using Affinity Column

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All the chemicals used in this study were of reagent grade and purchased from Sigma. The Affinity column (His-Trap FF) and the AKTA-Prime purification system were bought from GE Healthcare. The SX20 Stopped-Flow and SDS–PAGE and Western-Blot apparatus were obtained by AppliedPhotophysics and BioRAD, respectively.

Del Prete S., Bua S., Supuran C.T, & Capasso C. (2020). Escherichia coli γ-carbonic anhydrase: characterisation and effects of simple aromatic/heterocyclic sulphonamide inhibitors. Journal of Enzyme Inhibition and Medicinal Chemistry, 35(1), 1545-1554.

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Protein Purification and Characterization

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IPTG and antibiotics were purchased from Sigma. The affinity column (His-Trap FF) and molecular weight markers were from GE Healthcare. All other chemicals used in this study were of reagent grade. The AKTA-prime purification system was purchased by GE Healthcare. The SX20 Stopped-Flow was obtained from Applied Photophysics, while the SDS–PAGE apparatus was procured by BioRAD.

Del Prete S., Angeli A., Ghobril C., Hitce J., Clavaud C., Marat X., Supuran C.T, & Capasso C. (2020). Sulfonamide Inhibition Profile of the β-Carbonic Anhydrase from Malassezia restricta, An Opportunistic Pathogen Triggering Scalp Conditions. Metabolites, 10(1), 39.

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Reduction of S-mycothiolated GapDH

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GapDH, Mrx1 and Trx were reduced before the assays with 10 mM DTT for 30 minutes at room temperature. Excess of DTT was removed by desalting with Micro Biospin 6 columns. Pre-reduced GapDH (25 µM) was pre-incubated with 10-molar excess of MSH at 37 °C for 5 min, then 100-fold molar excess of H₂O₂ was added and the mixture was incubated at 37 °C for 5 min. Excess of H₂O₂ and MSH were removed on a PD-10 desalting column (GE Healthcare). The NADPH consumption during the de-mycothiolation reactions was monitored spectrophotometrically at 340 nm and 37 °C, using argon-flushed 50 mM Hepes/NaOH, pH 8, 500 mM NaCl, 1 mM EDTA. For the reduction of S-mycothiolated GapDH by the Trx pathway, we used 2 µM Trx, 5 µM Trx-reductase and 250 µM NADPH in a Spectramax 340PC plate reader (Molecular Devices). For the reduction of S-mycothiolated GapDH by the Mrx1 pathway, we used 20 nM Mrx1, 5 µM MSH, 5 µM MSSM reductase and 250 µM NADPH in SX-20 stopped flow (Applied PhotoPhysics). After 5 min pre-incubation of this mixture at 37 °C, 60 µM mycothiolated GapDH was added to initiate the reaction. Three technical and experimental replicates were performed.

Hillion M., Imber M., Pedre B., Bernhardt J., Saleh M., Loi V.V., Maaß S., Becher D., Astolfi Rosado L., Adrian L., Weise C., Hell R., Wirtz M., Messens J, & Antelmann H. (2017). The glyceraldehyde-3-phosphate dehydrogenase GapDH of Corynebacterium diphtheriae is redox-controlled by protein S-mycothiolation under oxidative stress. Scientific Reports, 7, 5020.

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Stopped-flow Kinetics of CAZ Binding

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CAZ binding kinetics were assayed using an SX20 stopped flow (Applied Photophysics) by tryptophane fluorescence, with an excitation wavelength of 280 nm, a 305 nm lower cutoff emission filter and a 0.2 mm excitation pathlength. Enzyme and substrate were mixed 1:1 at a final enzyme concentration of 1 μM, and the final substrate concentration was varied between 100 and 1,200 μM. Binding was assayed at 25 °C and in 0.1 M phosphate buffer (pH 7.2) supplemented with 50 mM NaHCO₃ (Sigma Aldrich). Binding was recorded on a log timescale, and rates were obtained by global fitting of the observed biphasic curves to a double-exponential decay (equation (3)).

Fluorescence = A_{1} \times \exp^{- k_{1} * t} + A_{2} \times \exp^{- k_{2} * t} + c

where k₁ and k₂ are the observed binding rates and A₁ and A₂ are the amplitudes of the two signals with an offset of c. To enable global fitting, k₁ and k₂ were fitted to a linear equation each (equations (4) and (5)), where k_1,on, k_2,on, k_1,off and k_2,off were shared between all datasets.

k_{1} = k_{1, on} \times c (CAZ) + k_{1, off}

k_{2} = k_{2, on} \times c (CAZ) + k_{2, off}

Fröhlich C., Bunzel H.A., Buda K., Mulholland A.J., van der Kamp M.W., Johnsen P.J., Leiros H.K, & Tokuriki N. (2024). Epistasis arises from shifting the rate-limiting step during enzyme evolution of a β-lactamase. Nature Catalysis, 7(5), 499-509.

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Burst Kinetics of Enzymatic Reactions

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Room-temperature burst kinetics were obtained under burst-phase conditions using an SX20 stopped flow (Applied Photophysics) by monitoring substrate depletion by absorbance at 260 nm. Enzyme and substrate were mixed 1:1 at 25 °C and in 0.1 M phosphate buffer (Sigma-Aldrich, pH 7.2) supplemented with 50 mM NaHCO₃ (Sigma-Aldrich). Burst kinetics were assayed at final enzyme concentrations of 10 μM and final substrate concentrations varying between 50 and 400 μM (equation (1)).

\frac{P}{E_{0}} = v_{steady} \times t - (v_{steady} - v_{burst}) \times (1 - e^{- k \times t}) / k

Catalytic parameters (k_cat, K_M and k_cat/K_M) were determined under burst-phase and steady-state conditions using CAZ (Δξ = −9,000 M⁻¹ cm⁻¹) at 260 nm by measuring the initial enzymatic reaction rate in an Epoch plate-reader (Biotek). Burst phase rates were determined at 4 °C, and steady-state parameters were determined at 25 °C. Reactions rates were obtained in at least duplicates at a final enzyme concentration of 1 μM (final assay volume of 100 μl). Ultraviolet-transparent 96-well plates (Corning) were used. Assays were performed in 0.1 M phosphate buffer (Sigma-Aldrich, pH 7.2), supplemented with 50 mM NaHCO₃ (Sigma-Aldrich).

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Kinetic Isotope Effects Measurement

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Kinetic isotope effects (KIE) were determined at 25 °C using an SX20 stopped flow (Applied Photophysics) from burst kinetics obtained at 400 μM CAZ. KIEs were calculated from the ratio of the rate in 80% D₂O (Sigma-Aldrich; k_D) and water (k_H; equation (2)).

KIE = \frac{k_{H}}{k_{D}}

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Pre-equilibrium Fluorescence Kinetics

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Pre-equilibrium fluorescence measurements were performed on an SX20 stopped-flow (Applied Photophysics), using 1:1 vol:vol mixing ratio, at 25 °C. Excitation wavelengths of 280 or 295 nm were selected with a monochromator, with slit widths of 0.5 mm and emission wavelengths above 320 nm (for tryptophan fluorescence) or 445 nm (for AEDANS fluorescence) were selected with cut-off filters. Data were collected over suitable time courses (varying from 50 ms to 1000 s) under pseudo-first-order conditions. The apparent rate constant was obtained by fitting data to a single exponential equation, with k_on determined from the gradient of k_app plotted against a range of excess protein concentrations.

Housden N.G., Webby M.N., Lowe E.D., El-Baba T.J., Kaminska R., Redfield C., Robinson C.V, & Kleanthous C. (2021). Toxin import through the antibiotic efflux channel TolC. Nature Communications, 12, 4625.

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Reversibility of Burst Phase Kinetics

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To assess the reversibility of the burst phase, 20 μM Q4 was pre-incubated with 400 μM CAZ, and mixed with a second batch of 400 μM CAZ after a delay of 1,000, 2,000 and 3,000 s in an SX20 stopped flow (Applied Photophysics) by monitoring substrate depletion by absorbance at 260 nm.

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Affinity Purification of His-Tagged Proteins

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All the chemicals used in this study were of reagent grade and purchased from Sigma. The Affinity column (His-Trap FF) and the AKTA-Prime purification system were bought from GE Healthcare. The SX20 Stopped-Flow was obtained by the Applied Photophysics. SDS–PAGE and Western blot apparatus were procured from BioRAD.

Del Prete S., De Luca V., Bua S., Nocentini A., Carginale V., Supuran C.T, & Capasso C. (2020). The Effect of Substituted Benzene-Sulfonamides and Clinically Licensed Drugs on the Catalytic Activity of CynT2, a Carbonic Anhydrase Crucial for Escherichia coli Life Cycle. International Journal of Molecular Sciences, 21(11), 4175.

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Purification and Analysis of Protein

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IPTG and antibiotic were purchased from Sigma, Affinity column (His-Trap FF), molecular weight markers from GE Healthcare. All other chemicals used in this study were of reagent grade. AKTA-Prime purification system was purchased by GE Healthcare. The SX20 Stopped-Flow was obtained by the AppliedPhotophysics, while SDS–PAGE apparatus was procured by BioRAD.

Del Prete S., Nocentini A., Supuran C.T, & Capasso C. (2020). Bacterial ι-carbonic anhydrase: a new active class of carbonic anhydrase identified in the genome of the Gram-negative bacterium Burkholderia territorii. Journal of Enzyme Inhibition and Medicinal Chemistry, 35(1), 1060-1068.

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