Application Notes

4 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. Methods Plasmids The p38α coding sequence originates from reverse transcription from mRNA from A549 cells and was fused on a mammalian expression vector behind a CMV promotor to the mNeongreen-6xHis coding sequence separated by a linker ESGSGS. Production of p38 in HeLa cells 3x106 HeLa cells were transfected with p38α- mNeonGreen-6xHis, and mNeonGreen-6xHis respectively, in individual T-75 fl asks. Cells were harvested a er 24 hours, when each pellet contained approximately 10x10 6 cells. Cells were lysed in 1 ml PBS-T buff er (137 mM NaCl, 2.5 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4; 0.05 % Tween-20) supplemented with protease inhibitors and disrupted with a Dounce homogenizer. Cell debris was removed by centrifugation at 20000 × g for 30 minutes. The lysate was diluted 1:10 in PBS-T containing protease inhibitors. Estimation of the concentration of His-tagged protein in cell lysate by MST A sixteen-point 1:1 serial dilution of cell lysate expressing p38α was prepared using non-transfected HeLa cell lysates in 10 µl of fi nal volume. 10 µl of 50 nM RED-tris-NTA dye were added to each reaction tube, resulting in a fi nal dye concentration of 25 nM. The reaction mixture was incubated for 30 minutes at room temperature and loaded into premium coated capillaries. Conclusion In this report, we demonstrate the applicability of RED-tris-NTA labeling of proteins in cell lysates to provide rapid and quantitative characterization of biomolecular interactions using MST. This approach can be useful in providing initial binding characterizations during the early stages of drug discovery, particularly for targets which are diff icult to purify, thus saving time and money. This application is not limited to protein-small molecule interactions as shown here and can also be applied to other systems, including protein-protein, protein-sugar or protein-nucleic acid interactions. Figure 3. MST traces and dose-response curve for the binding interaction between p38α-mNeonGreen-6xHis and SB203580. The concentration of p38α-mNeonGreen-6xHis in lysate is constant, while the concentration of the non-labeled SB203580 varies between 26.5 µM – 1.62 nM. A K d of 82 nM was determined for this interaction. Concentrations on the x-axis are plotted in µM. mNeonGreen fl uorescence was used for detection. The corresponding MST time traces are shown as an inset. n = 3

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