Application Notes

7 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. Material and Methods Labeling of His-tagged peptides and proteins in buffer The His6- and His10-tagged peptides and proteins (His6-IDH R132H, His6-pUL53, and His6-p38) were diluted to 200 nM in PBS-T buffer (137 mM NaCl, 2.5 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4; 0.05 % Tween-20). The RED-tris-NTA dye was diluted in PBS-T to 100 nM. Protein and the dye were mixed in 1:1 volume ratio and incubated for 30 min at room temperature. Production and Labeling of His6-pUL53 in the E. coli lysate The protein His6-pUL53(50–292) was produced in E. coli BL21(DE3) (Walzer et al., 2015). Bacterial cells were grown in TB medium in the presence of 45 μg /ml kanamycin at 37 °C to an A 600 of 0.6 before the temperature was lowered to 20 °C, and protein expression was induced with 0.25 mM isopropyl-D- thiogalacto-pyranoside overnight. Bacterial cells were harvested by centrifugation, resuspended in lysis buffer (50 mM phosphate buffer, pH 7.4, 300 mM NaCl) containing protease inhibitors, and disrupted by high pressure homogenization. In the same manner, lysis of bacterial cells containing no His6-pUL53 was produced for the dilution series in buffer. The lysate containing His6-pUL53 was diluted 1:10 in PBS-T and the RED-tris-NTA dye added at the final concentration of 50 nM. The mixture was incubated for 30 min at the room temperature. The ligand buffer was HEPES buffer (200 mM, 25 mM HEPES, 1 mM TCEP, pH 8.0). MST experiment The interactions with the dye or ligands with the peptide, His6-p38 and His6-pUL53 were measured in Standard treated capillaries, His6 IDH was measured in MST Premium coated capillaries. The measurements were performed in the PBS-T buffer. Before the MST measurements all samples were centrifuged for 10 min at 4 °C and 14000 g. For the binding studies His6-p38 and His6-IDH R132H were labeled with the RED-tris-NTA dye in PBS-T buffer in the ratio 1:2 (50 nM dye, 100 nM protein) for 30 min at the room temperature. The ligand dilution series (1:1) were prepared in PBS-T. The ligands for the binding studies were dissolved in PBS-T at two-fold concentration as indicated in the figures. Instrumentation and data analysis The measurements were performed on a NanoTemper Monolith™NT.115 instrument. Final dye concentration of 25 nM yielded the fluorescence intensity of around 300 counts at a LED power of 50 %. The samples were measured at MST power between 40 % and 60 % with a laser-on time of 30 s and a laser-off time of 5 s. The data were analyzed by MO Affinity Analysis So ware.

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