G1160

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.

PIP Strips were purchased from Echelon (Echelon Biosciences) and were used following the manufacturer's instructions. Briefly, GST and GST fusion proteins, expressed and purified following the protocol described above, were incubated with PIP Strips at the concentration recommended by Echelon. Control GST-fusion proteins, for testing binding to a specific phosphoinositide, were purchased from Echelon and used at the recommended concentration. To detect the bound protein to PIP Strips, the following reagents were used: mouse anti-GST antibody, (1∶2000, Sigma #G-1160), anti mouse IgG conjugated with Horseradish peroxidase (HRP), (1∶5000, Sigma #A-9917); ECL Prime Western Blotting Detection Reagent (GE Healthcare).

Whole cell extracts were separated by using 10% SDS-polyacrylamide gels and the proteins were transferred to nitrocellulose membranes. Overproduced GST-SpPol4 and α-tubulin were detected using anti-GST (G1160, Sigma) and anti-tubulin (T6074, Sigma) monoclonal antibodies, respectively. Enhanced chemiluminescence detection (Amersham Biosciences) and anti-mouse IgGs coupled to horseradish peroxidase antibody (Amersham Biosciences) were used for protein visualization.