title: Immunoblotting with Peptide Antibodies: Differential Immunoreactivities Caused by Certain Amino Acid Substitutions in a Short Peptide and Possible Effects of Differential Refolding of the Peptide on a Nitrocellulose or PVDF Membrane.
abstract: Immunodetection using antibodies, e.g., Western blotting, is generally utilized to measure the amount of a certain protein in a protein mixture. For valid interpretation of results observed by immunodetection, strict attention must be paid to the factors affecting the immunoreactivities of the antibodies. We here describe the step-by-step procedures to demonstrate that substitution of certain amino acids in a peptide can cause remarkable differences in its immunoreactivity with antibodies against epitope tags in the immobilized peptide. Refolding of the peptide on the membrane in a way that masks the epitope to different degrees was the possible reason for their distinct immunoreactivities with the antibodies. The results in this chapter suggest that we need to interpret carefully the experimental results involving immunodetection.
authors.affiliation: Institute for Genome Research, University of Tokushima, Kuramotocho 3-18, Tokushima, 770-8503, Japan. tyamamo@genome.tokushima-u.ac.jp.|Faculty of Pharmaceutical Sciences, University of Tokushima, Tokushima, Japan. tyamamo@genome.tokushima-u.ac.jp.
authors.forename: Takenori
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Institute for Genome Research, University of Tokushima, Kuramotocho 3-18, Tokushima, 770-8503, Japan.|Faculty of Pharmaceutical Sciences, University of Tokushima, Tokushima, Japan.
authors.forename: Taisuke
authors.initials: T
authors.lastname: Matsuo
authors.affiliation: Faculty of Pharmaceutical Science, Suzuka University of Medical Science, Suzuka, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yamamoto
authors.affiliation: Institute for Genome Research, University of Tokushima, Kuramotocho 3-18, Tokushima, 770-8503, Japan.|Faculty of Pharmaceutical Sciences, University of Tokushima, Tokushima, Japan.
authors.forename: Ryohei
authors.initials: R
authors.lastname: Yamagoshi
authors.affiliation: Faculty of Pharmaceutical Science, Suzuka University of Medical Science, Suzuka, Japan.
authors.forename: Kazuto
authors.initials: K
authors.lastname: Ohkura
authors.affiliation: Biomarker Analysis Research Group, Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Japan.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Kataoka
authors.affiliation: Institute for Genome Research, University of Tokushima, Kuramotocho 3-18, Tokushima, 770-8503, Japan.|Faculty of Pharmaceutical Sciences, University of Tokushima, Tokushima, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Shinohara
mesh_terms: D019943:Amino Acid Substitution; D000906:Antibodies / Q000276:immunology*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D015151:Immunoblotting / Q000379:methods*; D010455:Peptides / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D058849:Protein Refolding
chemical_list: D000906:Antibodies; D000939:Epitopes; D010455:Peptides
keywords: Antibody; Immunodetection; Nitrocellulose membrane; PVDF membrane; Refolding; Western blotting