Kinetic ligand-binding measurements were performed using an Octet RED384 BLI instrument (fortéBio). Superstreptavidin (SSA) biosensors were loaded with biotinylated macrodomain protein and equilibrated for 120 sec in assay buffer (25 mM HEPES (pH 7.4), 100 mM NaCl, 0.01 % Tween 20). Association and dissociation were monitored for 240 sec each in assay buffer at 25 °C. GeA-69 was prepared as seven 1:1 serial dilutions starting from 10 µM. Binding to the reference sensors (no protein attached) was subtracted before calculations and data was processed using the fortéBio analysis software provided by the manufacturer.
Octet red384 bli instrument
Manufactured by Molecular Devices
The Octet RED384 BLI instrument is a high-throughput label-free detection system that utilizes Biolayer Interferometry (BLI) technology to monitor biomolecular interactions in real-time. The instrument is capable of simultaneously analyzing up to 384 samples, providing efficient and comprehensive data collection for a wide range of applications.
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Lab products found in correlation
2 protocols using octet red384 bli instrument
1
Kinetic Analysis of Macrodomain Protein Binding
2
Biolayer Interferometry for Protein-DNA Binding
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ssDNA-protein binding assays were performed on an Octet RED384 BLI instrument (ForteBio). 3’-biotin-TEG labeled poly-dT35 ssDNA was captured at a concentration of 5 µg/mL for 100 s on streptavidin sensors. For protein-protein binding assays, His-tagged proteins were captured at a concentration of 5 µg/mL for 100 s on NTA sensors, subtracting non-specific signals from reference sensors loaded with His-tagged maltose-binding protein (MBP). Each binding experiment was performed at least three times at 25 °C in buffer E supplemented with 0.2 mg/mL BSA (A2153, Sigma). Data were analyzed with Prism 9.4. A blank sample reference with buffer-only was subtracted from all curves. Affinities were determined by fitting the concentration dependence of the experimental steady-state signals, using the following equation Req = Rmax * [RPA]/(Kd + [RPA]).
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