Octet biosensor analysis and protein biotinylation

Biosensor analysis was performed on a Fortebio Octet RED384 instrument (Pall Life Sciences) using Ni-nitrilotriacetic (NTA) or streptavidin (SA) sensor by functionalizing of 50 µg/ml His-tagged talin head (talin 1, residues 1–405⁸⁵) or biotinylated MBP-tagged proteins, respectively. Phosphate-buffer (50 mM NaPO₃ and 150 mM NaCl, pH 7.2) was used in all steps as running buffer when using the Ni-sensors and the octet kinetic buffer (PBS containing 1% BSA and 0.1% Tween-20, 0.05% sodium azide) was used when using SA-sensors. Every time new sensors were used and prior to run, they were soaked into the running buffer for 30 min according to the manufacturer’s instructions. The temperature of 27 ˚C and a stirring speed of 1000 rpm was used throughout the experiments. Ni-sensors were chemically activated by immersing them in 0.05 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 0.1 M N-hydroxysuccinimide (NHS; ThermoFisher Scientific) in Milli-Q water, which enabled covalent coupling of immobilized proteins⁸⁵. 1 M ethanolamine pH 8.5 was used in quenching step for the covalent coupling approach. Biocytin (biotinyl-lysine at 10 µg/ml) was used in quenching step for SA-sensors. The binding of the GST-tagged paxillin constructs with talin head was measured at 20, 80, 320, 1250, and 5000 nM. In the same manner, the binding of the talin head with the biotinylated MBP-tagged paxillin fragments immobilized on the SA-sensor was measured using the same concentrations.

In the case of talin head binding to immobilized MBP-LIMs, streptavidin biosensor functionalized with biotinylated free MBP was used as a reference and the binding response obtained was subtracted from the measured binding between talin and MBP-LIMs to obtain a specific binding response.

In the case of interaction assays between talin head and GST-LIM constructs, binding of free GST on Ni-NTA biosensor-immobilized talin head was used as a reference, which was subtracted from the measured binding between various GST-LIM samples and immobilized talin to obtain a specific binding response.

The MBP-proteins were biotinylated using EZ-Link^TM NHS-PEG₄-Biotin (Thermo Scientific) according to the instructions of the manufacturer. The non-reacted NHS-PEG₄-Biotin was removed by dialysis and the concentration of the proteins was measured by BCA-kit (Thermo Scientific). The biotin incorporation was estimated using HABA (4′-hydroxyazobenzene-2-carboxylic acid; Thermo Scientific) method. The absorbance of the HABA-avidin solution was measured before and after adding the biotinylated sample at 500 nm using Envision UV/VIS (Perkin Elmer). The amount of biotin molecule per sample was then calculated using HABA calculator which is a freely available tool from Thermo Scientific webpage.