Peptide SPOT Synthesis and Interaction

Peptide SPOT synthesis [43] (link) was performed essentially as described in [44] (link). Briefly, cellulose membrane-bound peptides were prepared in an automated Spot synthesizer (MultiPep, Intavis AG Bioanalytical Instruments, Köln, Germany) using Fmoc derivatives of amino-acids (Novabiochem, Darmstadt, Germany). GST-nTRIP6 was expressed in Escherichia coli BL21 and purified using glutathione Sepharose 4B beads (GE Healthcare, Freiburg, Germany). After activation of the membranes with methanol the membrane-bound peptide arrays were blocked for 3 h in blocking buffer (2% milk powder and 5% sucrose in Tris-buffered saline (TBS), pH 8.0) and then incubated overnight at 4°C with 10 µg/ml purified GST-nTRIP6 in blocking buffer, which was then detected with an anti-GST antibody (G1160; Sigma-Aldrich, München, Germany), revealed by a horseradish peroxidase conjugated anti-mouse antibody (Sigma-Aldrich) and ECL. The QRALAKDLIVPRRP peptide, recognized by the anti-GST antibody, was used as a positive control.