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APPLICATION NOTE
©2018 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
Conclusion
The TRIC-optimized Monolith Protein Labeling Kit
RED-NHS 2nd Generation (Amine reactive) provides
improved performance when analyzing molecular
interactions by MST. Examples of three different
types of interactions: protein-small molecule, protein-
protein and protein nucleotide were shared in this
report.
The kit uses a fluorophore that is specifically
developed and optimized for TRIC detection
of binding interactions. Higher MST response
amplitudes and S/N ratios compared to a previously
released RED-NHS labeling kit are obtained with
the new kit reagents (summarized in Table 1). The
key advantages to the RED-NHS 2nd Generation
kit are improved detection success rate and less
optimization time required for assay development.
Material and Methods
Protein labeling
Protein labeling with both dyes was carried out
according to the user manuals of the respective
labeling kits (RED-NHS, cat#MO-L001 and RED-
NHS 2nd Generation, cat# MO-L011) . For p38α and
Hsp90, 100 µL of 20 µM protein solution was mixed
with 100 µL of 60 µM dye solution. For ß-lactamase
TEM1 point mutant variant P107A, 100 µL of 20 µM
protein solution was mixed with 100 µL of 120 µM
dye solution. The labeling reaction was carried out in
the kit's NHS labeling buffer, for 30 min in the dark at
room temperature. A erwards, a gravity flow column
included in the labeling kit was used to remove excess
dye, before samples were eluted in MST-T buffer
(50 mM Tris-HCl pH 7.8, 150 mM NaCl, 10 mM MgCl
2
,
0.05% Tween 20,).
Preparation of reaction mixtures for MST experiments
For the interaction of p38α with SB239063: SB239063
was stored in 100% DMSO at -20 °C. For the serial
dilution, the compound was diluted to 4 µM
using MST-T buffer. This solution was used for the
preparation of a 16 step 1:1 serial dilution with a final
volume of 10 µL, while making sure that the final
DMSO concentration remains constant throughout
the dilution series (final DMSO concentration was 2%).
MST-T buffer was used as assay buffer. Next, 10 µL
of 40 nM labeled p38α was added to each SB239063
dilution and the mixture was incubated for 30 min at
room temperature in the dark.
For the interaction of TEM1 with BLIP: BLIP was
diluted to a final concentration of 1 µM using MST-T
buffer. This solution was used for the 16-step 1:1 serial
dilution in MST-T buffer, with a final volume of 10 µL in
each titration step. Next, 10 µL of 10 nM labeled TEM1
was added to each dilution.
For the interaction of Hsp90 with ADP: ADP was
diluted to a final concentration of 2 mM using MST-T
buffer. This solution was used for the 16-step 1:1 serial
dilution in MST-T buffer, with a final volume of 10 µL in
each titration step. Next, 10 µL of 60 nM labeled Hsp90
was added to each dilution.
