Optiplate 96 hs

The assay was performed in 96-well plates (OptiPlate-96 HS, Perkinelmer). The wells were incubated for 15 min with 200 μl solution containing 20 mM Tris/HCl pH 7.2, 100 mM NaCl, 1 mg/ml BSA and 1 nM cpNanoLuc biosensor. Subsequently HSA was added to the chosen wells to a final concentration of 100 nM. The plate was washed with 20 mM Tris/HCl pH7.2, 100 mM NaCl and after adding of 0.25 μl the NanoLuc substrate to the well the luminescence was recorded at 445–470 nm. The plate was then washed six times with 200 μl assay buffer containing 20 mM Tris/HCl pH 7.2, 100 mM NaCl. After washing, 200 μl assay buffer was added to the plate’s wells with or without 100 nM HSA. Following 1 h incubation the luminescence was measured again following the addition of 0.25 μl NanoLuc substrate. The luminescence signal of plastic absorbed biosensor represented about 1% of the input material.

Rapamycin, tacrolimus, human α-amylase, human serum albumin, and BSA were purchased from Sigma-Aldrich. NanoLuc substrate Furimazine was purchased from Promega. The luciferase assay plate OptiPlate-96 HS were purchased from PerkinElmer.