Monoclonal 1- and 3-Phosphohistidine Antibodies: New Tools to Study Histidine Phosphorylation
| Autor: | John R. Yates, Magda Stankova, Aaron Aslanian, Li Ma, Jacques Mauger, Stephen Rush Fuhs, Alan Binnie, Tony Hunter, Fahad Al-Obeidi, Anna Zagórska, Greg Lemke, Jill Meisenhelder |
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| Rok vydání: | 2015 |
| Předmět: |
medicine.drug_class
Peptide Biology Monoclonal antibody Article General Biochemistry Genetics and Molecular Biology Phosphoglycerate mutase Tandem Mass Spectrometry medicine Animals Humans Histidine Spindle Poles Phosphorylation Centrosome chemistry.chemical_classification Biochemistry Genetics and Molecular Biology(all) Autophosphorylation Antibodies Monoclonal Phosphoramidate Molecular biology 3. Good health Blot Models Chemical chemistry Biochemistry Peptides Chromatography Liquid HeLa Cells |
| Zdroj: | Cell. 162(1):198-210 |
| ISSN: | 0092-8674 |
| DOI: | 10.1016/j.cell.2015.05.046 |
| Popis: | SummaryHistidine phosphorylation (pHis) is well studied in bacteria; however, its role in mammalian signaling remains largely unexplored due to the lack of pHis-specific antibodies and the lability of the phosphoramidate (P-N) bond. Both imidazole nitrogens can be phosphorylated, forming 1-phosphohistidine (1-pHis) or 3-phosphohistidine (3-pHis). We have developed monoclonal antibodies (mAbs) that specifically recognize 1-pHis or 3-pHis; they do not cross-react with phosphotyrosine or the other pHis isomer. Assays based on the isomer-specific autophosphorylation of NME1 and phosphoglycerate mutase were used with immunoblotting and sequencing IgG variable domains to screen, select, and characterize anti-1-pHis and anti-3-pHis mAbs. Their sequence independence was determined by blotting synthetic peptide arrays, and they have been tested for immunofluorescence staining and immunoaffinity purification, leading to putative identification of pHis-containing proteins. These reagents should be broadly useful for identification of pHis substrates and functional study of pHis using a variety of immunological, proteomic, and biological assays. |
| Databáze: | OpenAIRE |
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