Automated holdup assay and the protocol of binding intensity calculations have been benchmarked on a previously published data set. This data set consists of 210 interactions and non-interactions between 5 His-MBP-PDZ domain constructs and 42 biotinylated C-terminal peptides for which binding intensities had been obtained in a previous study using Surface Plasmon Resonance (BIAcore) 12 (link). Like in that study, normalized Response Units (RUs) obtained for the interaction experiments (see Supplementary Table 1 ) were used instead of dissociation constants to interpret binding intensities of measured interactions.
Uncorrected binding intensities obtained for crude protein samples using the holdup assay correlate well with the reference binding intensities determined using BIAcore (r=0.69). Better correlations can even be obtained when comparing input-corrected holdup assay binding data with the BIAcore data (r=0.76). This analysis indicates that input correction using standard peaks results in more reliable binding intensities (seeSupplementary Data 1 and 2 (folder (“benchmarkHU”) as well as Supplementary Table 1 for exported caliper data, parameters and binding intensities).
Uncorrected binding intensities obtained for crude protein samples using the holdup assay correlate well with the reference binding intensities determined using BIAcore (r=0.69). Better correlations can even be obtained when comparing input-corrected holdup assay binding data with the BIAcore data (r=0.76). This analysis indicates that input correction using standard peaks results in more reliable binding intensities (see
