Zeba spin plates

The assay conditions developed by Korkmaz et al. [27 (link)] were optimised to detect PR3/PRTN3 in urine samples. After thawing, urines were desalted using Zeba spin plates (Thermo Scientific, product number 1860080, 3747 N. Meridian road, Rockford, IL 61101 U.S.A). A 90 µL aliquot of Zeba cleaned urine was combined with reaction buffer to reach a final concentration of 50 mM HEPES buffer pH7.4, 750 mM NaCl, 0.05% IGEPAL CA-630. The reaction mixture was mixed for 2 min before aliquoting to the plate and incubating for an additional 1 h at 37 °C. The substrate, ABZ-VAD(nor)V RDRQ-EDDnp (Peptides International, SNP-3232-v) stock dissolved in 30% (v/v) N,N-dimethylformamide was diluted to 0.2 mM with 50 mM Hepes buffer, pH 7.4 and then added at a final concentration of 5 µM and the reaction was monitored at Ex/Em 320/420 nm at 37 °C for 90 min. The slopes of duplicate reactions for each sample were estimated in the 15–30 min linear reaction range after correction for any background fluorescence occurring in the same urine assayed without substrate.

For single affinity purification, the PDZome v2 library was expressed in the same conditions (strain, media, buffers) than for the bacterial lysate^{27 (link)} but at a bigger culture scale (24 ml per PDZ instead of 6 ml per PDZ). The production (24 ml) and Ni-affinity purification (800 μl of beads/PDZ) follow strictly the protocol described in ref. 66 (link). The elution volume of Ni-affinity purification was reduced from 4 ml^{66 (link)} to 2.5 ml/PDZ. 96 PDZ are produced in parallel and are purified in four blocks. At the end of the purifications of these blocks purified PDZs are desalted using Zeba Spin plates (Thermo scientific) in 50 mM Tris, pH 8.0, 300 mM NaCl, 10 mM imidazole. The purity and concentration of each His₆-MBP-PDZ were evaluated with a microfluidic capillary electrophoretic system (Caliper LabChip GXII, PerkinElmer, Waltham, Massachusetts) and adjusted to 4 µM by dilutions before freezing the library in 96-well plate for the holdup experiment.