Methods to uncover an antibody epitope in the KPI domain of Alzheimer's amyloid precursor protein for immunohistochemistry in human brain.
A novel polyclonal antibody (Ab993), specific for a KPI domain epitope of APP, was characterised for use in immunoprecipitation, Western blotting and immunohistochemistry. Conditioned medium from NTera2/D1 cells was used for immunoprecipitation and Western blots. Paraffin-embedded human brain sections were used for immunohistochemistry. The antibody recognised KPI-containing APP on Western blots after standard solubilisation but immunoprecipitation of soluble APP required reduction with 2-mercaptoethanol followed by alkylation of reduced sulphydryl bonds with sodium iodoacetate. Immunohistochemical staining of human brain sections was significantly enhanced by this pre-treatment. Microwaving of sections also increased immunolabelling, by a mechanism that was additive to reduction and alkylation. Incubation in 80% formic acid did not confer any enhancement of immunoreactivity. Ab993, applied with the methods reported here, is expected to be valuable in investigations of the pathogenesis of Alzheimer's disease to determine the source of the beta-amyloid peptide.
Campbell
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Biomedical Research Centre, Division of Biomedical Science, Sheffield Hallam University, Sheffield, UK.
Pearson
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RC
Parkinson
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D000544:Alzheimer Disease / Q000235:genetics*; D000595:Amino Acid Sequence / Q000235:genetics; D016564:Amyloid beta-Protein Precursor / Q000235:genetics* / Q000276:immunology*; D000906:Antibodies / Q000276:immunology*; D015153:Blotting, Western; D002460:Cell Line; D000939:Epitopes; D006801:Humans; D007150:Immunohistochemistry; D008623:Mercaptoethanol; D008969:Molecular Sequence Data; D010455:Peptides; D010940:Plant Proteins; D011233:Precipitin Tests; D014361:Trypsin Inhibitors / Q000235:genetics*
D016564:Amyloid beta-Protein Precursor; D000906:Antibodies; D000939:Epitopes; C053865:Kunitz-type protease inhibitor, plant; D010455:Peptides; D010940:Plant Proteins; D014361:Trypsin Inhibitors; D008623:Mercaptoethanol