Heme oxygenase 2 polyclonal antibody

The antiserum was raised to a synthetic peptide, sequence: H-E-T-L-E-D-G-F-P-V-H-D-G-K-G-D-M-R-K-C-NH 2 corresponding to amino acid residues 246-264, in amidated form, of the human HO-2 protein (McCoubrey et al., 1992) . Two amino acid substitutions (Phe252 –> Leu; Met261 –> Val) exist in the aligned region within rat HO-2, and four (Leu248 –> Val; Gly251 –> Arg; Phe252 –> Leu; Met261 –> Val) in rabbit HO-2. The peptide was conjugated at the C-terminal cysteine residue via a maleimido linkage to keyhole limpet haemocyanin. The antiserum has been purified by caprylic acid and ammonium sulphate precipitation, and by protein G affinity chromatography. Application: Heme oxygenase-2 is constitutively expressed in many tissues, but the protein levels are extremely variable. The use of enriched enzyme preparations is recommended for experimental analysis. This antibody has been shown to react strongly with HO-2 on Western blots of microsomal preparations of unstimulated rat brain (HP 9311), testis (HP 9312) and to a lesser extent with liver (HP 9314) . Strong bands have also been detected in extracts of whole rat brain and HepG2, a human hepatocellular carcinoma cell line (ATCC HB8065) . A single band at ~36kDa is observed at a primary antibody dilution of 1:2000 or above when detected using enhanced chemiluminescence. Antibody HC 3002 is believed to be wholly specific for HO-2 showing no cross-reactivity with HO-1 in all systems studied to date. The immunostaining is fully abolished by pre-adsorption of the antibody with cognate peptide (HP 9302) . The antibody has been used successfully in immunocytochemical studies of HO-2 localisation in human bronchial epithelium biopsy material. Fixatives: Several fixative solutions may be used. Aldehyde-combination fixatives (i.e. those containing formaldehyde and glutaraldehyde) usually give satisfactory results. Bouin and Susa fixatives containing 0.1-0.2% glutaraldehyde have been used satisfactorily.