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APPLICATION NOTE
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lysate expressing mNeonGreen-6xHis fusion protein
but lacking p38α. First, the His-tag was labeled with
RED-tris-NTA in cell lysate and the ligand SB203580
was titrated. As shown in Figure 2 (grey), binding
could not be detected, demonstrating that the
changes in the MST signal of RED-tris-NTA-labelled
p38α are a result of its specifi c interaction with
SB203580.
In addition, orthogonal MST experiments were
carried out in which the mNeonGreen fl uorescence
of the protein was detected using the Monolith
NT.115 system's blue detection channel. As shown in
Figure 3, clear binding was detected and the aff inity
of SB203508 for p38α-mNeonGreen-6xHis was
determined to be 82 nM, which strongly correlates
with the previous results. Interestingly, comparison of
the results with the fi rst experiment, where the RED-
tris-NTA labeled protein was tested (Figure 2), show
that the labeling with RED-tris-NTA resulted in a larger
binding amplitude and a highly-improved signal-to-
noise ratio.
Figure 2. MST traces and dose-response curve for the binding interaction between RED-tris-NTA labeled p38α and SB203580 in whole cell lysate.
The concentration of RED-tris-NTA labeled p38α-mNeonGreen-6xHis (red) or mNeonGreen-6xHis (grey) in lysate is constant, while the concentration
of SB203580 varies between 26.5 µM and 1.62 nM. A K
d
of 247 nM was determined for p38α-mNeonGreen-6xHis. The negative control employing
mNeonGreen-6xHis did not yield a binding curve. Concentrations on the x-axis are plotted in µM. RED-tris-NTA fl uorescence was used for detection. The
corresponding MST time traces are shown on the right. n = 3
