Yamamoto Takenori, Matsuo Taisuke, Yamamoto Atsushi, Yamagoshi Ryohei, Ohkura Kazuto, Kataoka Masatoshi, Shinohara Yasuo
Institute for Genome Research, University of Tokushima, Kuramotocho 3-18, Tokushima, 770-8503, Japan.
Faculty of Pharmaceutical Sciences, University of Tokushima, Tokushima, Japan.
Methods Mol Biol. 2015;1348:303-10. doi: 10.1007/978-1-4939-2999-3_26.
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.
使用抗体进行免疫检测，例如蛋白质印迹法，通常用于测量蛋白质混合物中特定蛋白质的含量。为了有效解释免疫检测观察到的结果，必须严格关注影响抗体免疫反应性的因素。我们在此描述逐步程序，以证明肽中某些氨基酸的取代可导致其与固定化肽上针对表位标签的抗体的免疫反应性产生显著差异。肽在膜上以不同程度掩盖表位的方式重新折叠是其与抗体具有不同免疫反应性的可能原因。本章的结果表明，我们需要仔细解释涉及免疫检测的实验结果。
