Anti gst monoclonal antibody

The immobilized peptides of 15-amino acids length, sequentially overlapping by 13 residues (2aa shift), representing the entire sequence of TbPEX11 or the N-terminal domains of three human PEX11 isoforms were synthesized on a cellulose membrane as described previously (Hilpert et al., 2007 (link); Neuhaus et al., 2014 (link)). The peptide array was first washed with ethanol for 10 min with gentle shaking followed by three washes with TBS (50 mM Tris, 137 mM NaCl, 2.7 mM KCl, adjusted to pH 8) for 10 min each. Further, the peptide array was incubated with a blocking buffer (TBS +3% BSA +0.05% Tween-20) for 2 h at room temperature (RT). The purified recombinant proteins GST-TbPEX19, GST-HsPEX19, or GST alone (10 mL of 1 µM solution prepared in blocking buffer) were incubated with the arrays for 1 h at 4°C. Then, the arrays were washed three times for 10 min at RT with TBS, and subsequently incubated with the anti-GST monoclonal antibody (Sigma, 1:2000) at RT for 1 h. Followed by three washes with TBS (10 min each), a secondary antibody (Horseradish peroxidase-coupled anti-mouse IgGs, 1:5,000 in blocking buffer) was applied, and the array was further incubated for 1 h at RT. After three washes with TBS, the array was scanned with chemiluminescence substrate (WesternBright Sirius) using Azure sapphire biomolecular imager.

After SDS-polyacrylamide (10 %) gel electrophoresis, the proteins were transferred onto Immobilon-P transfer membranes (Millipore, Billerica, MA, USA) using a Mini Protean II (Bio-Rad, Hercules, CA, USA) electro-blotting apparatus at 100 V for 1 h in 25 mM Tris/192 mM glycine buffer, pH 8.3 containing 20 % methanol. After a blocking step specific antigens were revealed with serum from a mange-infested rabbit serum diluted 1:200, a mange-infested chamois diluted 1:100, an anti-GST monoclonal antibody diluted 1:5,000 (SIGMA, Madrid, Spain), and a mix of a rabbit pre-immune serum (1:100) and a serum from a mange-free chamois (1:200) followed by the addition of the appropriate species-specific peroxidase-conjugated secondary antibody. The immunocomplexes were revealed using 4-chloro-1-naftol as substrate.