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APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
The reversibility of the classical Ni(II)-mono-NTA
interaction is advantageous for applications such
as immobilized metal affinity chromatography
and surface immobilization, because it enables full
recovery of the immobilized His-tagged biomolecule
by adding a competitor such as imidazole that
disrupts the Ni(II)-NTA -His-tag interaction. His-tagged
proteins are e.g., eluted from Ni-NTA columns with
buffers containing high concentrations of imidazole.
Therefore, the reversibility of labeling using RED-
tris-NTA needs to be considered working at low
concentrations, since it might result in dissociation of
the dye. Thus, we analyzed in detail whether common
buffer components interfere with the RED-tris-NTA
labeling of the His-tagged protein. We found that
buffer components like Mg
2+
, Ca
2+
, bovine serum
albumin and other proteins without His-tag do not
interfere with the labeling procedure. A summary of
common buffer components and their maximum
allowed concentration for the RED-tris-NTA labeling is
presented in the Table 1.
Compound
Maximum allowed
concentration
Histidine 1 mM
Imidazole 1 mM
EDTA 0.5 mM
TCEP 0.5 mM
DTT 5 mM
A B C
Figure 2: MST traces (top) and dose-response curves (bottom) of His6 peptide (A), His6-IDH R132H (B) and His6-p38α (C) protein towards RED-tris-NTA. The resulting
dose-response curves were fitted to a one-site binding model to extract K
d
values. MST experiments were performed at a LED and MST power of 40%. Fnorm =
normalized fluorescence
