Template Instrumentation for "Accurate Constant via Transient Incomplete Separation".

Area of Science:

• Biophysical Chemistry
• Analytical Chemistry
• Biochemistry

Background:

• Accurate Constant via Transient Incomplete Separation (ACTIS) is a novel method for measuring protein-small molecule complex dissociation constants (Kd).
• Previous in silico studies confirmed the conceptual accuracy of ACTIS, independent of fluidic system geometry.
• Potential instrumental inaccuracies due to signal drift were identified as a limitation.

Purpose of the Study:

• To assemble a physical ACTIS instrument and demonstrate the absence of run-to-run signal drifts.
• To confirm the ruggedness and reliability of the ACTIS method by building a second instrument.
• To establish a standardized fluidic system for reproducible ACTIS instrumentation.

Main Methods:

• Assembly of a physical ACTIS instrument with a fluidic system mimicking in silico models.
• Experimental validation of signal drift absence in the physical instrument.
• Parallel experiments using two independently assembled ACTIS instruments with identical samples.

Main Results:

• The physical ACTIS instrument exhibited no significant run-to-run signal drifts.
• Two independently built ACTIS instruments produced identical Kd values for the same samples.
• Differences in instrument geometry and separagrams did not impact Kd determination accuracy.

Conclusions:

• The developed fluidic system design ensures reliable and reproducible ACTIS instrumentation.
• The ACTIS method is robust and accurate for determining protein-small molecule binding affinities.
• This work provides a template for constructing dependable ACTIS devices for biophysical studies.