High-Throughput Protein-Peptide Interaction Assay

The holdup assay was performed on a Tecan freedom Evo200 robot with 384-well plates in singlicate for the three 11-mer PTEN variants and the 13-mer PTEN peptide as described in [31 (link),32 (link)]. Briefly, prior to interaction assay, 2.5 μL of streptavidin resin was saturated in each well with 20 μL of biotinylated PBM peptides (42 μM) and then washed twice with an excess of free biotin, while the reference resin was incubated only with biotin. Right before the holdup experiment, the PDZ library was spiked with an internal standard of lysozyme. Then, the biotin- or PBM-saturated resin was incubated, each in a distinct well of a 384-well plate, with complete cell lysates diluted so that the concentration of the tag-PDZ present in the crude extract is adjusted at 4 μM. After a sufficient time to reach complex equilibrium (15 min.), a fast and mild filtration step is performed and the tag-PDZ concentrations were measured by capillary electrophoresis instrument (LabChip GXII, PerkinElmer, Massachusets, USA). Standard markers were used to convert migration time into molecular weight on the LabChip software and inappropriate molecular weight markers were corrected or excluded.