Octet red 96 system

Full details of the developed assay, including a full methodology and data analysis, can be found in the validated protocol [56 (link)] and further data using this protocol [54 (link),55 (link)]. In summary, the BLI assay was performed on the Octet Red 96 system (ForteBio, Fremont, CA, USA). Venom (analyte) samples were diluted at 1:20 (a final experimental concentration of 50 µg/mL per well). Mimotope aliquots were diluted at 1:50 (a final concentration of 1 µg/mL per well). The assay running buffer was 1X DPBS with 0.1% BSA and 0.05% Tween-20. Prior to experimentation, Streptavidin biosensors were hydrated in the running buffer for 30–60 min, whilst on a shaker at 2.0 revolutions per minute (RPM). The dissociation of analytes occurred using a standard acidic solution glycine buffer (10 mM glycine (pH 1.5–1.7) in ddH₂O). Raw data are provided in Supplementary File S1. All data obtained from BLI on Octet Red 96 system (ForteBio) were processed in accordance with the validation of this assay [56 (link)]. The association step data (in triplicate) were obtained in an Excel.csv file extracted from raw outputs of the Octet Red 96 system and then imported into Prism8.0 software (GraphPad Software Inc., La Jolla, CA, USA) and graphs were produced.

All data obtained from BLI on Octet RED 96 system (ForteBio, Fremont, CA, USA) were processed in exact accordance to the validation of this assay [32 (link)]. The association step data (in triplicate) were obtained in an excel.csv file extracted from raw outputs of the Octet RED 96 system and then imported into Prism 7.0 software (GraphPad Software Inc., La Jolla, CA, USA) where area under the curve (AUC) calculations were made and graphs produced.
Phylogenetic trees were obtained from timetree.org and then further manually recreated using Mesquite software version 3.2 (http://mesquiteproject.org/). The obtained phylogenetic trees were then further analysed in RStudio (R Core Team, 2015) for all comparative analysis using the Ape package [61 (link)]. Heat-mapping of AUC values over the phylogenetic trees was achieved using the contMap function of the R package phytools [62 (link)].