Cortisol-based Fluorescent Sensor Conjugation

First, 50 μM
of purified LUCOS-base proteins were diluted in 1 mL 100 mM sodium
phosphate buffer (pH 7) and reduced with 5 mM tris(2-carboxyethyl)phosphine
(TCEP) for 1 h at room temperature (shaking incubator 500 rpm). Next,
to remove the excess of TCEP, the sample was loaded onto a PD-10 desalting
column (Cytvia) that was first equilibrated with 100 mM sodium phosphate
buffer (pH 7) with 25 μM TCEP. For the maleimide coupling reaction,
the reduced LUCOS-base protein was mixed with the cortisol-3-CMO-maleimide
in a 1:10 molar ratio (7.5 μM sensor protein with 75 μM
cortisol-3-CMO-maleimide) in 100 mM sodium phosphate buffer (pH 7,
with 5% DMSO). The reaction mixture was subsequently incubated for
2 h at room temperature (500 rpm). Next, a PD-10 desalting column
was used to remove excess, unreacted cortisol-3-CMO-maleimide and
exchange the reaction buffer with PBS (pH 7.2). Successful incorporation
of the analog was confirmed with Q-ToF LC-MS (Waters MassLynx v4.1),
using MagTran v1.03 for MS deconvolution (Figures 2b,c and S5). Proteins
were stored at −70 °C until use.