Autospot robot ass 222

Peptide libraries spanning the full length of APP were produced by automatic SPOT synthesis and synthesised on continuous cellulose membrane supports on Whatman 50 cellulose membranes using Fmoc-chemistry with the AutoSpot-Robot ASS 222 (Intavis Bioanalytical Instruments AG, Köln, Germany) as we have previously described^{50 (link)}. Arrays were incubated with recombinant human LRP6 (Fc-chimera, R&D systems) or total lysate from HEK293A cells overexpressing HA-tagged Vangl2. Binding was visualised using either an anti-HA tag (Abcam, ab9110) antibody or anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, DyLight 680 (Thermo Fisher, SA5-10138) and imaged on a Chemidoc system with Image Lab software (BioRad).

Peptide libraries were generated by automatic SPOT synthesis on Whatman 50 cellulose membranes using Fmoc (9-fluorenylmethyloxycarbonyl) chemistry with the Autospot-Robot ASS 222 (Intavis Bioanalytical Instruments, Koeln, Germany). The interaction of peptide spots with GST and GST-fused purified proteins by overlaying the cellulose membranes with 10 mg/ml recombinant protein was determined as described previously in detail [16 (link),34 (link),35 (link)]. Bound recombinant proteins were detected with specific primary antisera and complementary HRP-coupled secondary antibody as for immunoblotting.

Immobilised 25‐mer peptides were spotted onto cellulose membranes using an Autospot Robot ASS222 (Intavis®, Cologne, Germany) as previously described [47 (link)]. Arrays were subjected to in vitro SUMOylation Assay (Enzo) and subsequently immunoblotted using primary antibodies and HRP‐conjugated secondary antibodies. Enhanced chemiluminescence (ECL) was used to visualise arrays and positive spots indicated antibody detection.

Libraries of immobilized peptides were produced by automatic SPOT synthesis on continuous cellulose membrane supports (Whatman 50 cellulose membranes) using Fmoc (fluoren-9-ylmethoxycarbonyl) chemistry with the AutoSpot-Robot ASS 222 (Intavis Bioanalytical Instruments AG)65 (link). Individual peptide–cellulose complexes were solubilized and re-spotted on Celluspot slides for subsequent probing. Slides were primed in binding buffer (4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 5 mM MgCl₂, 1 mM DTT, 30 μM GDP and 1% (v:v) TX100) and blocked for 1 h in the same buffer supplemented with 5% (w:v) BSA. Slides were incubated for 2 h at room temperature with rat His-Gαi3 at 20 μg ml⁻¹ (∼0.5 μM) in the same buffer. After four washes, slides were sequentially incubated with primary (rabbit anti-Gαi3, 1: 250; 90 min) and secondary (goat anti-rabbit Alexa Fluor 680, 1:10,000; 60 min) antibodies. Images were acquired in an Odyssey infrared scanner (Li-Cor), processed using the Image J software (NIH) and assembled for presentation using Photoshop and Illustrator softwares (Adobe).

Peptide libraries were produced by automatic SPOT synthesis (51 (link)). They were synthesized on continuous cellulose membrane supports on Whatman 50 cellulose membranes using Fmoc (9-fluorenylmethyloxycarbonyl) chemistry with the AutoSpot-Robot ASS 222 (Intavis Bioanalytical Instruments, Berlin) (52 (link)). The SNAP25 and CSPα arrays consisted of 15-mer peptides (200 peptides each), with all possible amino acid substitutions within a 10-amino acid stretch (sequences and serial substitution shown in Fig. 1). The dried membranes were submerged in 100% ethanol for 2 min, washed briefly with distilled water, and then incubated with blocking buffer: 5% (w/v) milk in PBS-T (PBS containing 0.02% Tween) for 2 h at room temperature. Then, after a brief washing with PBS-T, overnight incubation with the ARzD17-His protein (500 nm in PBS-T) took place at 4 °C. Membranes were then washed extensively with PBS-T, incubated with mouse His₆ tag antibody (1/2,000 dilution in PBS-T) for 1 h at room temperature, washed again with PBS-T, incubated with mouse secondary antibody (1/10,000 dilution in PBS-T), and after extensive washes with PBS-T containing 0.2% Tween, ARzD17-His-bound peptides were visualized using a LI-COR infrared scanner. Spots were quantified using Image Studio software (version 2.0), with an area of the blot containing no peptide assigned as background.

Peptide libraries were produced by automatic SPOT synthesis and synthesized on continuous cellulose membrane supports on Whatman 50 cellulose membranes using Fmoc-chemistry with the AutoSpot-Robot ASS 222 (Intavis Bioanalytical Instruments, Köln, Germany) as previously described.^{17 (link)} Phosphorylation of peptide array peptides by constitutively active GSK3 (14-306, Millipore UK) was undertaken as previously described.^{18 (link)} Phosphorylation was detected using a phospho-serine specific antibody (ab9332, Abcam, UK).