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A P P L I C A T I O N N O T E
Protein expression and purification
All membrane proteins were either expressed in
HEK293 or T.ni. cells and purified via a C-termi-
nal Rho1D4 Tag utilizing HighSpec Rho1D4 Mag-
Beads (Cube Biotech Order number: 33205). The
detailed purification protocol ("Membrane Pro-
tein Solubilization Protocol with Copolymers")
can be accessed via the Cube Biotech Website.
In short: Cells were thawed in buffer with pro-
tease inhibitor cocktail, lysed via sonication.
Cell debris were separated via centrifugation,
then the membrane fraction was purified via
ultracentrifugation. Membrane pellets were sol-
ubilized via NativeMP
TM
Copolymer (best fit pre-
determined experimentally) and subsequent-
ly bound to Rho1D4 MagBeads for 3h. Purified
target protein was eluted via HighSpec Rho1D4
Peptide (Order number: 16201), concentrated to
350 µl and run over Superose 6 10/300GL Size
Exclusion Chromatography. Fractions were an-
alysed via Prometheus Panta, SDS-PAGE and
Western Blot. Fractions of interest were pooled
and used for analysis.
Monolith X experiments
Covalent labeling of lysine residues of each
membrane protein was performed using com-
mercially available Cy5 analogues with a de-
gree of labeling of approximately 0.5-1. For the
Monolith X measurement, each ligand dilution
(16 of 1:1 dilutions) was mixed with 40 nM la-
beled protein, which led to a final concentration
of protein of 20 nM. A er 30 min incubation, the
samples were loaded into Monolith Capillaries
(cat# MO-K022, NanoTemper Technologies).
Spectral Shi measurements were performed
at 25°C using the Monolith X instrument (Nan-
oTemper Technologies). Instrument parameters
were adjusted to auto-excitation power and
medium IR laser power. Spectral Shi (Ratio
670/650 nm) or TRIC (ΔFnorm) of all ligand con-
centrations were calculated automatically with-
in the MO.Control so ware (v2.6.3, NanoTemper
Technologies GmbH). Data of independently pi-
petted measurements were analyzed using the
MO.Control so ware (v2.6.3, NanoTemper Tech-
nologies GmbH).
Material and Methods
