1 1
A P P L I C A T I O N N O T E
Figure 8: (A) DLS analysis of copolymer nanodisc-stabilized NTCP (cyan) and LHBsAg (purple). The cumulant
analysis reported the polydispersity index (PDI) of the sample NTCP and LHBsAg to be 0.20 ± 0.04 and 0.16 ± 0.04
respectively. The size distribution analysis reported the hydrodynamic radius (rH) to be 6.57 ± 0.22 nm and 5.80 ±
0.29 nm for the NTCP and LHBsAg nanodiscs. (B). Binding of nanodisc-stabilized LHBsAg to nanodisc-stabilized
NTCP (cyan). The fluorescently labeled NTCP was used at a constant concentration of 20 nM with var ying concen-
trations of the LHBsAg in 20 mM HEPES pH 7.5 and 150 mM NaCl. Fluorescence intensity at 670 was used to get TRIC
response (on time 2.5s) for interaction analysis yielding the Kd of 967 nM with S/N ratio of 12.78. Error bars repre-
sent the standard deviation of 3 independent measurements and with a confidence of 95%, Kd is within 669 - 1396
nM. The black data shows the negative control using the labeled NTCP mixed with a dilution series of a non-specific
membrane protein.
The interaction studies between two full-length membrane proteins presents a significant
challenge for researchers. The utilization of nanodiscs obviates the necessity to consider
the detergents required for each membrane protein. In this case study, the interaction is
observed exclusively through TRIC detection. TRIC and Spectral Shi are essentially two or-
thogonal biophysical methods based on two biophysical principles. When investigating an
unknown interaction or working with an unconfirmed assay condition for the first time, com-
bining two biophysical principles in a single measurement on a single instrument represents
a great advantage, enhancing the success of interaction analysis.
0
2
4
6
8
10
12
14
16
Relative
probability
1 10 100 1000
Radius [nm]
NTCP2P3final
L-HBsAg1P4final
10 100 1000
Radius [nm]
NTCP2P3final
L-HBsAg1P4final
925
926
927
928
929
930
931
932
Fnorm
[‰]
1x10
-10
1x10
-9
1x10
-8
1x10
-7
1x10
-6
1x10
-5
1x10
-4
Ligand Concentration [M]
(A) (B)
