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APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
Interaction measurements in crude bacterial cell lysate
The broad application range of the RED-tris-NTA
labeling was demonstrated by direct labeling of a
target protein, His6-pUL53, in crude E. coli lysate
followed by the measurement of the interaction
between the labeled target protein and its binding
partner pUL50. Both proteins pUL53 and pUL50
form the core nuclear egress complex of human
cytomegalovirus (HCMV) (Walzer, Egerer-Sieber et
al., 2015). His6-pUL53 could be efficiently labeled
with RED-tris-NTA directly in the lysate as shown in
Figure 4. The dilutions series of the lysate containing
His6-pUL53 was prepared in mock lysate which was
void of any His-tagged protein. The concentration
of His6-pUL53 was estimated based on the previous
experiences with the purification yields. To determine
the binding affinity of pUL50 towards His6-pUL53,
His6-pUL53 was labeled either directly in the lysate or
a er purification in buffer. For this experiment, PBS
T was used as labeling buffer and the dilution series
was prepared in HEPES buffer. For the purified His6-
pUL53 a K
d
value of 1.20 ± 0.45 μM was determined.
The measurement in lysate yielded a K
d
value of
1.78 ± 0.24 μM. Determined K
d
values did not differ
significantly between both experimental approaches,
highlighting the robustness and reproducibility of the
RED-tris-NTA labeling system.
A B
Figure 3: The MST traces (top) and dose-response (bottom) curves for the binding interactions between the RED-tris-NTA labeled
His6-IDH R132H (A), His6-p38α (B) and their ligands. Measurements were performed at LED power 50 % and MST power 80 %.
Fnorm = normalized fluorescence
