3
Fig. 2: Binding of NT-647-labeled TEM1 to BLIP
protein. In the MST experiment, we kept the
concentration of fluorescently-labeled TEM1-WT
constant at 10 nM, while the concentration of BLIP was
varied. After 10 min, an MST analysis was performed
(n = 3). A) Binding of BLIP-WT to NT-647-labeled
TEM1-WT. A K
d
of 3.5 nM ± 0.6 nM was determined for
this interaction. B) Binding of BLIP-W112A to NT-647-
labeled TEM1-WT. A K
d
of 474 nM ± 76 nM was
determined for this interaction. C) Binding of BLIP1-
W150A to NT-647-labeled TEM1-WT. A K
d
of 1750 nM
± 220 nM was determined for this interaction. All MST
time traces are shown above the binding curves.
In a second experiment we have used a
fluorescence fusion protein in order to
characterize the binding of Ypet-BLIP (Fig. 3)
to TEM1-WT or TEM1-R243A. Fig. 4
Illustrates the Ypet-BLIP TEM1 complex.
Fig. 3: The Ypet-BLIP construct. A 6-His tag and Ypet
were fused to the N-terminus of BLIP with a five amino
acid linker between them. A schematic of the Ypet-BLIP
TEM complex is at the bottom.
The concentration of Ypet-BLIP-WT was kept
constant at 15 nM, while the concentration of
TEM1-WT or TEM1-R243A was varied. After
a short incubation, the samples were loaded
into MST standard treated glass capillaries
(K002, NanoTemper technologies GmbH,
Germany) and MST analysis was performed.
A K
d
of 4.7 nM +/- 0.75 nM was determined
for the binding of Ypet-WT BLIP to WT TEM
(Fig. 4A), which correlates nicely with the
binding affinity determined using NT-647-
conjugated TEM1-WT. A 50-fold weaker
affinity was determined for the binding of
Ypet-BLIP-WT to TEM1-R243A (Fig. 4B).
These results confirm published SPR-data,
A
B
C
