Abstract
Phospho-specific primary antibodies are used in immunohistochemistry (IHC) to detect phosphorylated sequences in proteins, in some cases they may also cross-react with non- or de-phosphorylated sequences. To rule out nonspecific staining, and to determine that the staining pattern is specific it is necessary to employ a so-called absorption control: phospho-specific primary antibodies are first incubated with phospho-peptide immunogen to block antibody binding sites, and this mixture is applied to tissue sections. If the antibody pre-blocked with cognate immunogen does not produce tissue staining, then the antibody is considered specific. However, if the staining does occur, it indicates that the antibody is nonspecific. The drawback of doing absorption by mixing the peptide with the antibody is that in solution such peptide-antibody complexes can dissociate unblocking the antibody which becomes capable of binding to cell and tissue targets, producing unwanted staining. To overcome this problem, we have developed a simple absorption control technique allowing for efficient blocking of phospho-specific antibodies with phospho-peptides immobilized on magnetic beads. This technique allows for sequestration of peptide-antibody complex from the incubation mixture eliminating the risk of un-blocking primary antibodies via their dissociation from the blocking peptide.
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Kalyuzhny AE (2009) The dark side of the immunohistochemical moon: industry. J Histochem Cytochem 57:1099–1101
Mandell JW (2003) Phosphorylation state-specific antibodies: applications in investigative and diagnostic pathology. Am J Pathol 163:1687–1698
Mandell JW (2008) Immunohistochemical assessment of protein phosphorylation state: the dream and the reality. Histochem Cell Biol 130:465–471
Chen HF, Xie LD, Xu CS (2010) The signal transduction pathways of heat shock protein 27 phosphorylation in vascular smooth muscle cells. Mol Cell Biochem 333:49–56
Kaminska B (2009) Molecular characterization of inflammation-induced JNK/c-Jun signaling pathway in connection with tumorigenesis. Methods Mol Biol 512:249–264
Baba HA, Stypmann J, Grabellus F, Kirchhof P, Sokoll A, Schäfers M et al (2003) Dynamic regulation of MEK/Erks and Akt/GSK-3beta in human end-stage heart failure after left ventricular mechanical support: myocardial mechanotransduction-sensitivity as a possible molecular mechanism. Cardiovasc Res 59:390–399
Acknowledgments
We wish to thank Ernesto Resnik at R&D Systems, Inc. for his assistance with magnetic beads and MagCellect protocol.
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Schwartz, D., Grahek, M., He, Y., Wang, W., Nguyen, J., Kalyuzhny, A.E. (2017). Using Phospho-Peptides Immobilized on Magnetic Beads for Absorption Control in Immunohistochemistry. In: Kalyuzhny, A. (eds) Signal Transduction Immunohistochemistry. Methods in Molecular Biology, vol 1554. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6759-9_15
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DOI: https://doi.org/10.1007/978-1-4939-6759-9_15
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6757-5
Online ISBN: 978-1-4939-6759-9
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