Hrp conjugated anti gst antibody

SPOT synthesis of peptide arrays on cellulose membranes were performed using a MultiPep automated peptide synthesizer (INTAVIS Bioanalytical Instruments AG, Germany) as previously described [55 (link)]. After blocking the cellulose membranes in TBST with 5% nonfat dry milk, peptide interactions with GST or GST fusion proteins were tested by overlaying the membranes with 1 μg/ml of recombinant protein for 2 hr at room temperature. Filters were washed in TBST, and bound proteins were detected with HRP-conjugated anti-GST antibody (1:5000; clone RPN1236; GE Healthcare).

GST or GST-ATG8 proteins were expressed (from GST-ATG8 (pAL) plasmids) in E. coli BL21 (DE3) plysS cells (Agilent) in LB medium supplemented with 50 µg/ml kanamycin. Expression was induced by addition of 0.5 mM IPTG at OD₆₀₀ = 0.6 and cells were incubated at 25 °C overnight or at 37 °C for 5 h. Harvested cells were lysed in 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.1% Triton X-100, 0.4 mM AEBSF and 15 µg/ml Benzamidine. Fusion protein was batch-adsorbed onto Glutathione-Sepharose 4B beads (GE Healthcare). After five washes with wash buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 0.4 mM AEBSF and 15 µg/ml benzamidine) fusion proteins were eluted in 50 mM Tris pH 8.0, 2 mM L-glutathione reduced, 0.4 mM AEBSF and 15 µg/ml benzamidine.
A MultiPep or ResPep SL automated synthesizer (INTAVIS Bioanalytical Instruments AG, Germany) was used for SPOT synthesis of peptide arrays on cellulose membranes.⁷⁰ (link) After blocking membranes in TBST with 5% nonfat dry milk, peptide interactions with GST or GST fusion proteins were tested by overlaying the membranes with either 1 µg/ml (mutational peptide array scan) or 2 µg/ml of recombinant protein (all other peptide arrays) for 2 h at room temperature. Membranes were washed in TBST, and bound proteins were detected with HRP-conjugated anti-GST antibody (1:5000, GE Healthcare, RPN1236).