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TECHNICAL NOTE
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Methods and materials
Proteins and compounds were obtained from Sigma-Aldrich: CAII (Cat# C2522),
Furosemide (Cat# F4381), Acetazolamide (Cat# A6011) and Benzenesulfonamide (Cat#
108146).
Labeling of CAII
CAII was fluorescently labeled with the RED-NHS 2nd Generation dye (Cat# MO-LO11).
Briefly, CAII was dissolved in labeling buffer (130 mM NaHCO
3
pH 8.2, 50 mM NaCl, 0.1%
Pluronic® F-127) to a concentration of 70 µM. Next, protein aggregates were removed by
centrifugation (15,000 rpm, 4 °C, 20 min) and the protein concentration of the supernatant
was adjusted to 40 µM with labeling buffer. Subsequently, 50 µL of 40 µM CAII was mixed
1:1 with 40 µM ZnCl
2
(in labeling buffer) to obtain a 20 µM CAII/20 µM ZnCl
2
solution. The
solution was mixed 1:1 with 60 µM dye to a final volume of 200 µL and incubated at room
temperature in the dark. A er 30 minutes the unreacted, free dye was separated from the
labeled protein using a gravity flow column equilibrated with dilution buffer (50 mM Tris-
HCl pH 7.8, 150 mM NaCl, 10 mM MgCl
2
, 0.1% Pluronic F-127). Finally, the flow-through
was centrifuged again for 15 minutes at 15,000 rpm and 4 °C and the supernatant was
transferred to a fresh tube.
MST measurements
For all MST measurements, the sample buffer used was 50 mM Tris-HCl buffer pH 7.8,
supplemented with 150 mM NaCl, 10 mM MgCl
2
, 0.1% Pluronic® F-127 and 0.5% DMSO.
Samples to be measured were transferred to premium coated capillaries (Cat# MO-K025)
and loaded onto the sample tray and into the Monolith instrument. MST measurements
were carried out with the Binding Affinity module in the MO.Control 2 so ware with the
temperature set to 20 °C. % LED and MST power varied by experiment and are specified in
the Results.
