Application Notes

2 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. His-tag labeling in cell lysate was achieved using the RED-tris-NTA dye, which is designed to specifi cally bind polyhistidine tags with high aff inity (Tschammer et al., 2016). In addition to the His-tag, p38α was fused and co-expressed with an mNeonGreen protein, which allowed to validate the specifi city of the labeling approach and to verify the precision of binding data by measuring MST of the fusion protein in response to the inhibitor. Results To ensure an optimal protein-to-dye ratio for subsequent His-tag labeling, MST experiments were conducted for estimating the concentration of p38α in the lysate. Concentration determination was achieved by titrating the lysate containing p38α-mNeonGreen- 6xHis against the RED-tris-NTA dye. By using the previously published K d value for the aff inity of RED- tris-NTA dye towards a His-tagged purifi ed p38α (2.3 nM) (Tschammer et al., 2016), the p38α concentration in the cell lysate was estimated to be between 50 and 100 nM. Subsequently, p38α was directly labeled in cell lysate following the RED-tris-NTA labeling protocol. A er serial dilution of SB203580 in PBST and addition of unpurifi ed, RED-tris-NTA-labelled p38α, the aff inity of the interaction was measured by following binding- induced changes in the MST signal. As shown in Figure 2, a clear binding-induced change in MST was detected, yielding a K d of 247 nM, which is comparable to previously published results (Cuenda et al, 1995). To test for specifi city of the binding signal, negative control experiments were performed using whole cell how MicroScale Thermophoresis (MST) can be used to perform biophysical interaction studies with unpurifi ed proteins, thereby saving valuable resources and time in a variety of research projects. The interaction between p38α, a serine/threonine protein kinase belonging to the mitogen-activated protein kinase (MAPK) family (Dominguez et al. 2005), and a well-characterized small-molecule antagonist SB203580 (Davies et al., 2000) was used as a model system to test interaction studies by MST using purifi cation-free labeling in cell lysate. p38α is known to play a major regulatory role in proinfl ammatory pathways and therefore is a key target for compound screening in the pharmaceutical industry. The interaction between purifi ed p38α and SB203580 was successfully characterized previously using MST (Saxena et al., 2011). In this study, we show that the same interaction can be quantifi ed in an entirely purifi cation-free system. Figure 1. Schematic illustration of RED-tris-NTA labeled p38α bound to a small molecule inhibitor. (PDB 3ZS5)

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