7
A P P L I C A T I O N N O T E
Figure 4: (A). DLS analysis of copolymer nanodisc-stabilized P2X4. The cumulant analysis reported the polydisper-
sity index (PDI) of the sample to be 0.24 ± 0.02. The size distribution analysis reported the hydrodynamic radius (rH)
of the P2X4 nanodisc to be 10.81 ± 0.45 nm. (B). Binding of antagonist 5-BDBD to nanodisc-stabilized P2X4. The fluo-
rescently labeled P2X4 was used at a constant concentration of 20 nM with varying concentrations of the 5-BDBD in
20 mM HEPES pH 7.4, 150 mM NaCl, and 0.005% Tween 20. Ratios of the fluorescence intensities at 670 and 650 nm
were used for interaction analysis yielding the Kd of 4.29 µM with S/N ratio of 14. Error bars represent the standard
deviation of 2 independent measurements and with a confidence of 95%, Kd is within 2.87-6.42 µM.
Following confirmation of the sample quality, the interaction analysis was conducted using the
Monolith X as described in the method section. The Cy5 analogue-labeled P2X4 nanodisc was main-
tained at a constant concentration of 20 nM, while the concentration of 5-BDBD was varied between
2 nM and 45 µM (Figure 4B). The Kd was determined by analyzing the Spectral Shi signal - ratio of
fluorescence intensities at 670 and 650 nm - yielding a value of 4.29 µM which aligns with the pub-
lished data (24,25). It is also noteworthy that the signal-to-noise ratio (S/N) of the Kd fit in Figure 4B
has a value of 14. The S/N value is a measure of the binding response amplitude relative to the noise
level of the replicates. An S/N value greater than 12 indicates an excellent condition for detecting
the binding.
0
2
4
6
8
10
Relative
probability
1 10 100 1000
Radius [nm]
1.3
1.32
1.34
1.36
1.38
670nm
/
650nm
1x10
-9
1x10
-8
1x10
-7
1x10
-6
1x10
-5
1x10
-4
Ligand Concentration [M]
(A) (B)
