Application Notes

Purification-free labeling in whole cell lysate and binding characterization by MST

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5 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. Since the aff inity of the RED-tris-NTA dye towards the 6xHis-tag is in the low nM to pM range and therefore well below the concentration of RED-tris-NTA, the binding curve will display a "kink" at the point where equimolar concentrations of dye and His-tagged protein are present (Figure 4). Since there is a single binding site for the RED-tris-NTA dye, one can assume that dye and protein concentrations are equivalent at the "kink", so that the concentration of the protein at the start of the dilution can be calculated. In this experiment, saturation of the binding curve was not reached, suggesting that the concentration of the protein in the lysate was less than 2-fold above the concentration of the dye (less or equal to 50 nM, data not shown). Labeling of p38α-mNeonGreen-6xHis The RED-tris-NTA dye was diluted in PBS-T to 100 nM. Diluted lysate and dye were mixed in 1:1 volume ratio and incubated for 30 minutes at room temperature. Figure 4. Simulation of a binding curve for an interaction between RED-tris-NTA dye and a 6xHis-tagged protein. The concentration of the target (RED-tris-NTA) is above the K d . Therefore, the binding curve shows a saturation "kink" at the concentration where the dye is fully saturated by 6xHis-tagged protein. MST experimental protocol SB203580 stock solution (2.65 mM, 100 % DMSO) was diluted 1:50 in PBS-T buff er. The dilution series was prepared by mixing 10 µl of 53 µM SB203580 with an equal volume of PBS-T buff er containing 2 % DMSO in 1:1 steps, resulting in a precise 16-step dilution series of the ligand. 10 µl of lysate containing the fl uorescent target protein was added to each reaction tube, decreasing the fi nal DMSO concentration to 1 % and the highest ligand concentration to 26.5 µM, whereas the concentration of supernatant was constant in each tube. All interactions were measured in Monolith NT.115 MST Premium Coated Capillaries at room temperature with an MST-on time of 20 seconds and an MST-off time of 5 seconds. The interaction of p38α- mNeonGreen and SB203580 was measured at 100% LED and High MST power. The interaction of RED-tris- NTA-labeled p38α-mNeonGreen-6xHis and SB203580 was measured at 20 % LED and High MST power. The data were collected by the MO.Control So ware and analyzed using the MO.Aff inity Analysis So ware. Instrumentation Measurements were performed using a NanoTemper ® Technologies Monolith® NT.115 system.

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