Generation of phospho-specific Hop1 antibodies

Polyclonal antibodies against the Hop1 phospho-T318 and phospho-S298 were obtained as following: The α-pT318 polyclonal antibody [Cambridge Research Biochemicals] was obtained by immunising two rabbits with the antigenic [C]-Ahx-ASIQP-[pT]-QFVSN where C is a cysteine residue added at the N-terminus of the peptide, Ahx is aminohexanoicacid and pT is a phosphorylated threonine residue. Upon bleeding, antibodies were purified through two affinity columns (each followed by a purification pass), the first adsorbing antibodies that bind to non-phosphorylated peptides and the second adsorbing the phospho-specific antibodies to pT318. The specificity of the antibody was tested using ELISA (enzyme-linked immunosorbent assay) analysis. The polyclonal phospho-specific antibody against phosphorylated serine residue 298 [Eurogentec] was obtained by immunising four guinea pigs with the antigenic peptide [C]-PQNFVT-[pS]-QTTNV, where C represents a cysteine added at the N-terminus of the peptide and pS is a phosphorylated serine residue. The α-pS298 antibody was purified in a similar manner to the α-pT318 antibody.