Technical Notes

2 TECHNICAL NOTE ©2018 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. manual. Next, labeled p38α was added to a dilution series of the small inhibitory compound SB203580 at a final concentration of 50 nM in low binding plates. The mixture was incubated at room temperature for 30 minutes prior to the MST experiment. Triplicate measurements were performed with a Monolith NT.115 in Premium Capillaries (MO-K025) at medium MST power and 40 % LED. The system was operated with the latest version of the MO. Control so ware (v 1.6) that has updated analysis settings optimized for TRIC-sensitive dyes. Additionally, acquired data sets were analyzed by MO.Affinity Analysis so ware (v 2.3) at 1.5 seconds MST on-time. Figure 1 shows the MST traces (le panel) as well as the binding curves (right panel) for experiments performed using both versions of the RED-tris-NTA labeling kits. For the interaction of SB203580 and p38α-RED-tris-NTA 2nd Generation, a K d of 9.15 ± 2.77 nM was determined, whereas an affinity of 8.66 ± 5.23 nM was derived for SB203580 and p38α-RED-tris-NTA (see Table 1). just like the RED-tris-NTA dye, binds efficiently and selectively to polyhistidine-tags and has minimum impact on the biochemical and physicochemical attributes of the protein or peptide. Additionally, the high affinity and selectivity of the dye for His-tags enables the labeling of target proteins even in crude cell lysates. Combining the advanced properties of the 2nd Generation dye with the merits of a site specific and purification-free labeling approach further broadens the range of interactions accessible with MST and simplifies the way to better results. Results and Discussion His-tagged, inactive p38α kinase was labeled using either the conventional His-Tag Labeling Kit RED- tris-NTA (MO-L008) or the novel His-Tag Labeling Kit RED-tris-NTA 2nd Generation (MO-L018). For both labeling approaches, p38α was labeled in phosphate- buffered saline supplemented with 0.05 % Tween-20 (PBS-T) for 30 minutes according to the labeling RED-tris-NTA 2nd Generation RED-tris-NTA Ligand Concentration [M] MST on time [s] 0 5 10 15 20 0.90 0.95 1.00 ] - [ e c n e c s e r o u l F e v i t a l e R 1.0E-11 1.0E-10 1.0E-09 1.0E-08 1.0E-07 1.0E-06 1.0E-05 1.0E-04 -8 -6 -4 -2 0 ] ‰ [ m r o N F ∆ Figure 1: MST traces (le panel) and dose response curves (right panel) for SB203580 titrated against p38 -RED-tris-NTA 2nd Generation ( ) or p38 -RED- tris-NTA ( ) at medium MST power. An MST on-time of 1.5 seconds was used for analysis and K d values of 9.15 nM ± 2.77 nM and 8.66 ± 5.23 nM were derived (n = 3 independent measurements, error bars represent the standard deviation). Corresponding amplitudes and S/N ratios are compared in Table 1.

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