Red tris nta 2nd generation

To test the binding affinity of maize RALF peptides with the corresponding interactors, MST assays were carried out by using a Monolish NT.115 (NanoTemper Technologies). His-tagged RALF proteins were labeled with red dyes with the His-Tag labeling kit RED-tris-NTA 2nd Generation (NanoTemper Technologies) according to the manufacturers’ recommendation. His-tagged RALF proteins were diluted to 200 nm in PBST buffer (137 mm NaCl, 2.5 mm KCl, 10 mm Na₂HPO₄, 2 mm KH₂PO₄, 0.05% Tween-20 at pH 7.4). A volume of 100 μL protein solution (200 nm) was mixed with 100 μL dye solution (100 nm) and incubated at room temperature for 30 min. After centrifugation, 20 nm dye-labeled proteins were mixed with a serial dilution of interactors (ZmFERL1/4/7/9, ZmPEX2/4). Samples were loaded into glass capillaries (NT.115 Standard Treated Capillaries) and measured at 40% MST power and 10% LED power. Recorded data were analyzed with MO. Affinity Analysis Software v2.3 (NanoTemper Technologies).

The direct binding of the Z4 and Z4P compounds to IRE1 protein was measured using MST. IRE1 recombinant protein was labelled using RED-Tris-NTA fluorescent dye (RED-Tris-NTA 2^nd Generation, NanoTemper, Munich, Germany; # MO-L018). For the labelling step, 100 μL of 20 nM protein solution is mixed with 100 μL of 10 nM RED-Tris-NTA dye in PBST buffer (PBS with 0.05% Tween-20) and incubated for 30 min at RT. The protein–dye mixture was centrifuged for 10 min at 4°C and 15 000xg. For measurement of direct binding, the compounds were analyzed in a 16-point dilution series mixed in a 1:1 ratio with the labelled protein in PBST buffer. The assay was performed in standard Monolith NT.115 Capillaries (NanoTemper; #MO-K022), and all measurements were performed at 60% MST power and 60% excitation power using the Monolith NT.115 Pico machine (NanoTemper). The dissociation constant (Kd) was calculated by taking the average of triplicate normalized fluorescence data using NANOTEMPER analysis software (MO.Affinity Analysis v2.3). The normalized values were converted to fraction-bound data, and the resulting binding curves are plotted using GRAPHPAD PRISM software (GraphPad Software).