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TMX-1000 Titration Module for the KinTek SF-300X Stopped-Flow

Equilibrium titration data are often as important as kinetic data in working to establish a reaction mechanism and can help to decipher stopped-flow signals. The KinTek TMX Titration Module fills the need for an advanced, automated system to titrate reactions at equilibrium. Taking advantage of the smooth drive of the SF-300X servo motor, the TMX unit allows the slow addition of titrant from a 10, 25 or 50 µL syringe into a 200 µL sample that is constantly being stirred. Simultaneously, the system records absorbance or fluorescence (or both, up to 3 channels), to provide a complete titration curve with 1000 points, in less than a minute. The volume of addition of titrant and time of data collection are fully programmable to cover a range of concentrations and times required for the reaction to come to equilibrium.

TMX-1000 data

Fluorescence Titration. This figure shows a fluorescence titration of 100 nM enzyme, showing a 20% change in signal. The smooth curve (buried under the points) is the best fit to the quadratic equation with a dissociation constant of 0.25 ± 0.01 µM. The titration required only 200 μL of solution and data collection was completed in 300 seconds.

Data can be fitted by nonlinear regression to either a Hyperbolic or a Quadratic equation. Accordingly, the titration can provide both the dissociation constant for the ligand and a measurement of the active site concentration. The system software automatically computes the concentration of titrant and corrects the signal for dilution at the option of the operator. Simply load the sample, attach the syringe containing the titrant, and press “Begin Titration.” Data are displayed immediately and can then be fitted using the system software or exported to a text file for importing into any one of numerous PC graphics programs.

The TMX allows the full functionality of the SF-300X data collection including up to 3 channels of simultaneous data recording. For example, two channels of fluorescence can be recorded to compute fluorescence anisotropy as a function of titrant concentration. The system is simple and easy to use, in keeping with the philosophy that drives KinTek Corporation; namely, to build instruments that encourage researchers to do the right experiments with ease, while providing the highest quality data and rigorous quantification. The TMX comes complete with thermostatted cell holder, a 5 × 5 mm cuvette, miniature stir bar and stirring motor drive system. Simply attach it to your SF-300X Stopped-Flow motor drive and begin collecting data! Note: this system will not work with pneumatic stopped-flow systems, as it requires a precision motor drive.

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