Sx 20 stopped flow apparatus

Manufactured by Applied Photophysics

Sourced in United Kingdom

The SX-20 stopped-flow apparatus is an analytical instrument used to study rapid chemical reactions. It is designed to mix two or more liquid samples and monitor the resulting changes in the solution over a short time scale, typically in the millisecond range. The core function of the SX-20 is to provide a fast and accurate way to measure the kinetics of a wide range of biochemical and chemical processes.

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Igor pro, by Wavemetrics (2 mentions) Origin 9, by OriginLab (2 mentions) Kintek explorer, by Applied Photophysics (1 mentions) Prism 9, by GraphPad (1 mentions) Pro kii software, by Applied Photophysics (1 mentions) Prism 7, by GraphPad (1 mentions)

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Stopped-flow apparatus (20 citations) SX-20 micro-volume stopped-flow apparatus (2 citations)

11 protocols using sx 20 stopped flow apparatus

Characterizing Ras-Nucleotide Interactions

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All stopped-flow experiments were performed in a SX-20 stopped-flow apparatus (Applied Photophysics) at 25 °C in a buffer consisting of 20 mM Hepes pH 7.5, 100 mM NaCl, 1 mM TCEP, 2 mM MgCl₂ (excitation with 360 nm LED, emission detection with a 420 nm cut-off filter).
For competition experiments, binding of 1 µM nucleotide free wild-type KRas (referred to as KRas_WT in the following) or KRas_1–169 G12C C51S C80L C118S (referred to as KRas_G12C) to 1 µM mantdGDP or 1 µM mantdGDPβMe in the absence of or the presence of 0.5 µM, 1 µM and 3 µM of competing nucleotides (GDP, GDPβMe, acyclovir or SML-8-73-1) was assessed and the resulting progress curves were globally fit using KinTek Explorer (the corresponding files are available as a supplement and can be opened with KinTek Explorer^{28 (link)} (http://kintekcorp.com/software/).
SOS catalyzed nucleotide exchange on 200 nM KRas:mantdGDP or KRas:mantdGDPβMe was characterized by exchange with 50 µM GDP in the presence of different concentrations of SOS, the resulting progress curves were fit with a single exponential equation and observed rate constants were plotted against the SOS concentration to obtain k_cat/K_M from the slope of the linear fit (Fig. 3A).
Allosteric activation of SOS was tested by mixing 10 µM KRas:mantdGDP with 1 µM SOS/1 mM GppNHp (Fig. 3C).

Müller M.P., Jeganathan S., Heidrich A., Campos J, & Goody R.S. (2017). Nucleotide based covalent inhibitors of KRas can only be efficient in vivo if they bind reversibly with GTP-like affinity. Scientific Reports, 7, 3687.

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Monitoring Aminoacyl-tRNA Interaction and Translocation Kinetics

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To monitor the time course of aminoacyl-tRNA interaction with the A site of the ribosome, we rapidly mixed 0.1 μM Phe-tRNA^Phe(Prf16/17)•EF-Tu•GTP ternary complex with 0.4 μM initiation ribosome complexes programmed with MF-mRNA at 20°C. To study the pre-steady-state kinetics of translocation, we rapidly mixed 0.06 μM ribosome pre-translocation complexes containing deacylated tRNA_i^fMet in the P site and fluorescently labeled fMet-Phe-tRNA^Phe(Prf16/17) (46 (link)) in the A site with 2 μM EF-G in the presence of 1 mM GTP at 37°C. Fluorescence was recorded using SX-20 stopped-flow apparatus (Applied Photophysics). Proflavine fluorescence was excited at 460 nm and measured after passing through a KV495-nm cut-off filter. Light-scattering experiments were carried out at 430 nm, followed by detection at a 90° angle without the cut-off filter. Samples were rapidly mixed in equal volumes (60 μl). Time courses were obtained by averaging 5-to-7 individual transients. Data were evaluated by fitting with a two-exponential function with characteristic time constants (k_app1, k_app2), amplitudes (A₁, A₂) and final signal amplitude (F_∞) according to equation F = F_∞ + A₁*exp(–k_app1*t) + A₂*exp(–k_app2*t), where F is the fluorescence at time t. All calculations were performed using the GraphPad Prism 9.3.1 software (GraphPad Software, Inc).

Paranjpe M.N., Marina V.I., Grachev A.A., Maviza T.P., Tolicheva O.A., Paleskava A., Osterman I.A., Sergiev P.V., Konevega A.L., Polikanov Y.S, & Gagnon M.G. (2022). Insights into the molecular mechanism of translation inhibition by the ribosome-targeting antibiotic thermorubin. Nucleic Acids Research, 51(1), 449-462.

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Measuring Oxygen Dissociation Kinetics

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Determination of O₂ dissociation rates was completed following previously described procedures [15 (link),25 (link),26 (link)]. PccGCS H237A/K238A was reduced in an anaerobic chamber using sodium dithionite, buffered exchanged into Buffer D, removed from the anaerobic chamber, and then mixed with oxygenated Buffer D. Rapid mixing with an equal volume of sodium dithionite solution (50 mM Tris pH 7.0, 50 mM NaCl, 1 mM DTT, 5 mM sodium dithionite) occurred within a SX20 stopped-flow apparatus (Applied Photophysics). O₂ dissociation was monitored with a diode array detector. Global fitting was completed with Pro-KII software (Applied Photophysics) and additional fitting analysis handled with Igor Pro (Wavemetrics, Portland, OR).

Walker J.A., Wu Y., Potter J.R, & Weinert E.E. (2020). π-Helix controls activity of oxygen-sensing diguanylate cyclases. Bioscience Reports, 40(2), BSR20193602.

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Aptamer-Protein Interaction Kinetics

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To measure the kinetics of aptamer-protein interactions an SX20 stopped-flow apparatus was used (Applied Photophysics, UK). All reactions were performed in SELEX Buffer (pH 7.4). Aptamers, PfR6 and PfE3, were pre-annealed to the probe^FAM as described above and called from herein, PfR6^FAM and PfE3^FAM. Fluorescent measurements were carried out at 22 ˚C using a monochromatic light source (470 nm) powered with 10 mA. Emitted fluorescence was measured with photomultipliers set at 320 V after passing a long-pass filter with a cut-off of 515 nm essentially as described in [35 (link)]. Equal volumes (60 μL) of each reactant were rapidly mixed and fluorescence changes over time were measured. Typically, one solution contained aptamer (0.25 μM), probe^FAM (0.2 μM) while the second solution was composed of 1 μM HMG-box^540QPf or Hs, quenching derivatives of their respective proteins labeled with Atto-540Q. Between 7 to 10 replicates were measured. Every single measurement acquired 1,000 data points in a logarithmic sampling mode. Apparent rate constants were estimated by non-linear regression with exponential equations using Prism 7.02 (Graphpad Sofware, USA). Averaged rate constants were calculated as described in [36 (link)].

Joseph D.F., Nakamoto J.A., Garcia Ruiz O.A., Peñaranda K., Sanchez-Castro A.E., Castillo P.S, & Milón P. (2019). DNA aptamers for the recognition of HMGB1 from Plasmodium falciparum. PLoS ONE, 14(4), e0211756.

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Stopped-flow Fluorescence Assay for dUTPase

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Fluorescence stopped-flow measurements were carried out at 20 ^oC using an SX-20 stopped-flow apparatus (Applied Photophysics, UK) as described previously24 (link). Equal volumes (50 μl) of dUTPase enzyme and dUTP solutions were mixed and 8 traces were recorded and averaged for each time course. Under single turnover conditions, a triple exponential equation was fitted to the averaged traces to determine the catalytic constants based on Tóth et al. JBC24 (link). For the determination of binding rate constants, the ligand titration was performed under pseudo-first order conditions. The observed rate constants for the two binding steps described previously (collision complex formation and isomerisation24 (link)) were determined by fitting double exponential equations. Where exponential equation for the first part of the time course could not been fitted due to the large signal loss in the dead time, the K_d was estimated by plotting the amplitudes of the fluorescence decrease against ligand concentration followed by fitting a hyperbole.

Szabó J.E., Takács E., Merényi G., Vértessy B.G, & Tóth J. (2016). Trading in cooperativity for specificity to maintain uracil-free DNA. Scientific Reports, 6, 24219.

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Stopped-flow Analysis of Tropomyosin-Troponin Regulation

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An SX-20 stopped-flow apparatus (Applied Photophysics) was used. K_B, the equilibrium constant between the blocked and closed states, was determined as previously described (Barrick et al., 2019 (link); Clippinger et al., 2019 (link)). WT data are from Clippinger et al. (2019) (link). At both low (pCa 9) and high calcium (pCa 4), 5 µM phalloidin-stabilized pyrene actin, 2 µM tropomyosin, 2 µM troponin, and 0.04 U/ml apyrase were rapidly mixed with 0.5 µM S1 myosin and 0.04 U/ml apyrase. Performed at 20°C, each experiment was the average of at least three separate mixes, and the data were fit by a single exponential curve. K_B was calculated from

\frac{k_{o b s} (- C a^{2 +})}{k_{o b s} (+ C a^{2 +})} = \frac{K_{B}}{1 + K_{B}} .

The reported K_B is the average of at least three different experiments. The P value was calculated from a two-tailed Student’s t test.
The rate of ADP release from myosin bound to regulated thin filaments (20°C) was measured as previously described. (Clippinger et al., 2019 (link)) The average and standard deviation of the rate of at least four experiments was calculated and the P value was derived using a two-tailed Student’s t test.

Clippinger S.R., Cloonan P.E., Wang W., Greenberg L., Stump W.T., Angsutararux P., Nerbonne J.M, & Greenberg M.J. (2021). Mechanical dysfunction of the sarcomere induced by a pathogenic mutation in troponin T drives cellular adaptation. The Journal of General Physiology, 153(5), e202012787.

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Stopped-Flow Analysis of Protein-Liposome Binding

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The association kinetics of protein binding to liposomes was obtained using an SX20 stopped-flow apparatus (Applied Photophysics). The starting concentration of the liposomes was maintained at 1 mM (DOPC with 20 mol % Ni–NTA-DGS) and the protein concentration was varied from 50 nM to 4 μM. Upon initiation of the stopped flow, the two solutions were mixed in an observation chamber having a short deadtime (∼ms) and the fluorescence was monitored by a photomultiplier tube (PMT) with a 495 nm long-pass filter to eliminate any scattering of the excitation light (485 nm) from reaching the detector. All measurements were performed at room temperature, and the curve fitting was performed using Igor Pro (Wavemetrics, Portland, OR) as detailed in the SI (Figure S4).

Raghunath G, & Dyer R.B. (2019). Kinetics of Histidine-Tagged Protein Association to Nickel-Decorated Liposome Surfaces. Langmuir : the ACS journal of surfaces and colloids, 35(38), 12550-12561.

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Rab15 and Rab3A Kinetic Measurements

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Rep15: mant-GppNHp Rab15 kinetic measurements were performed in Rab buffer (20 mM Hepes 8.0, 50 mM NaCl, 2 mM MgCl₂ and 2 mM DTE) using a SX-20 stopped-flow apparatus (Applied Photophysics) at 25 °C. The experiments were performed using the signal from the methylanthraniloyl group of mant-GppNHp/mant-dGppNHp, excited with a 365 nm and emission was detected through a 420 nm cutoff filter. Change in the fluorescence polarization of mant signal of 1 µM Rab15_mant-GppNHp (post-mix) was observed with an increasing amount of Rep15. All stopped-flow results that were analyzed are averages of 6–8 individual traces. Single/double exponential functions were fit using the Origin9 software (OriginLab).
For the Rep15: mant-GppNHp kinetics measurements, a change in the fluorescence intensity of mant signal of 0.5 µM Rab3A_mant-GppNHp (post-mix) was observed with an increasing amount of Rep15 (Buffer: 20 mM Hepes 8.0, 50 mM NaCl, 1 mM MgCl₂ and 2 mM DTE).

Rai A., Singh A.K., Bleimling N., Posern G., Vetter I.R, & Goody R.S. (2022). Rep15 interacts with several Rab GTPases and has a distinct fold for a Rab effector. Nature Communications, 13, 4262.

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Rab Kinetic Measurements by Stopped-Flow

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bMERB: Rab kinetic measurements were performed in Rab buffer (20 mM Hepes 7.5, 50 mM NaCl, 1 mM MgCl₂, and 2 mM DTE) using a SX-20 stopped-flow apparatus (Applied Photophysics) at 25 °C. For bMERB:mantGppNHp Rab8a_1-176 kinetic measurements, the experiments were performed using the signal from the methylanthraniloyl group of mantGppNHp, the mant group was excited with a 360 nm LED, and emission was detected through a 420 nm cutoff filter.
For bMERB:CH kinetic measurement, the experiments were performed in CH buffer using the signal from the Cy3 group of the N-terminal Cy3 labeled CH domain and was excited with a 535 nm LED and emission was detected through a 570 nm cutoff filter. For Rab8a dissociation experiments, CH buffer (20 mM Hepes 7.5, 150 mM NaCl, and 2 mM DTE) was used. All stopped-flow results that were analyzed are averages of 6–8 individual traces. Single exponential functions were fit using the Origin9 software (OriginLab).

Rai A., Bleimling N., Vetter I.R, & Goody R.S. (2020). The mechanism of activation of the actin binding protein EHBP1 by Rab8 family members. Nature Communications, 11, 4187.

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Transient Kinetic Measurements of Dbp5-ATP Dynamics

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Transient kinetic measurements were performed with an Applied Photophysics SX2.0 stopped-flow apparatus thermostatted at 25 ± 0.1 °C [13] (link), [14] (link), [15] (link), [30] (link), [66] . The time courses shown are either of raw, unaveraged traces or the average of 2–3 traces. The concentrations stated are final after mixing. Uncertainties are reported as standard errors in the fits unless stated otherwise. The following minimal reaction scheme with corresponding fundamental rate (k) and equilibrium constants (K, defined by ratio of corresponding k values) was used for analysis and modeling[13] (link), [14] (link), [15] (link), [66] : where H is Dbp5, T is ATP, D is ADP, and P_i is inorganic phosphate. We assume that product release is sequential with P_i preceding ADP. Several processes (e.g., nucleotide binding) are likely to be associated with multiple transitions (e.g., binding followed by an isomerization of the Dbp5 nucleotide complex) but are omitted for simplicity [8] (link), [13] (link), [14] (link), [15] (link), [66] .

Wong E.V., Cao W., Vörös J., Merchant M., Modis Y., Hackney D.D., Montpetit B, & De La Cruz E.M. (2016). Pi Release Limits the Intrinsic and RNA-Stimulated ATPase Cycles of DEAD-Box Protein 5 (Dbp5). Journal of Molecular Biology, 428(2Part B), 492-508.

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