Generation of PMAP-36 antibody.

Rabbit polyclonal antibody against synthetic PMAP-36 peptide (GRFRRLRKKTRKRLKKIGKVLKWIPPIVGSIPLGCG-amide) was generated at Biogenes (Berlin, Germany). Twenty-five micrograms of PMAP-36 peptide was synthesized by Fmoc chemistry at a purity of >80% with quality control by high-performance liquid chromatography (HPLC) and mass spectrometry. Five milligrams of PMAP-36 was conjugated to limulus polyphemus hemocyanin (LPH) with 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester and used to immunize two rabbits. Immunization followed a schedule with several boosts during several months and enzyme-linked immunosorbent assay (ELISA) titer testing of antisera, after which antisera from both rabbits were collected after final bleeding. Hundreds of milliliters of pooled antiserum was purified by affinity chromatography on a PMAP-36 coated CNBr-Sepharose column. Monospecific IgG was then eluted from the column with 0.2 M glycine–HCl buffer containing 250 mM NaCl (pH 2.2), neutralized with 2 M Tris-HCl (pH 7.5) and filtered (pore width, 0.45 μm) to remove any remaining debris. To conserve the antibody, 0.1% ProClin 300 was added.