Abstract
Immunoblotting is a ubiquitous immunological technique that aids in detecting and quantifying proteins (including those of lower abundance) and their posttranslational modifications such as phosphorylation, acetylation, ubiquitylation, and sumoylation. The technique involves electrophoretically separating proteins on an SDS-PAGE gel, transferring them onto a PVDF (or nitrocellulose) membrane and probing with specific antibodies. Here we describe an immunoblotting technique for detecting cellular phosphohistidine, a labile posttranslational modification, by optimizing experimental conditions such that the labile phosphohistidine signal is conserved throughout the experiment.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to NC sheets: procedure and applications. Proc Natl Acad Sci U S A 76:4350–4354
Glenney JR, Zokas L, Kamps MP (1988) Monoclonal antibodies to phosphotyrosine. J Immunol Methods 109:277–285
Heffetz D, Fridkin M, Zick Y (1989) Antibodies directed against phosphothreonine residues as potent tools for studying protein phosphorylation. Eur J Biochem 182:343–348
Fujimuro M, Sawada H, Yokosawa H (1994) Production and characterization of monoclonal antibodies specific to multi-ubiquitin chains of polyubiquitinated proteins. FEBS Lett 349(2):173–180
Kee JM, Muir TW (2012) Chasing phosphohistidine, an elusive sibling in the phosphoaminoacid family. ACS Chem Biol 7(1):44–51
Attwood PV, Piggott MJ, Zu XL, Besant PG (2007) Focus on phosphohistidine. Amino Acids 32(1):145–156
Wei YF, Matthews HR (1991) Identification of phosphohistidine in proteins and purification of protein-histidine kinases. Methods Enzymol 200:388–414
Fuhs SR, Meisenhelder J, Aslanian A, Ma L, Zagorska A, Stankova M et al (2015) Monoclonal 1- and 3-phosphohistidine antibodies: new tools to study histidine phosphorylation. Cell 162:198–210
Fuhs SR, Hunter T (2017) pHisphorylation: the emergence of histidine phosphorylation as a reversible regulatory modification. Curr Opin Cell Biol 45:8–16
Hindupur SK, Colombi M, Fuhs SR, Matter MS, Adam K (2018) The protein histidine phosphatase LHPP is a tumour suppressor. Nature 555(7698):678–682
Srivastava S, Panda S, Li Z, Fuhs SR, Hunter T, Thiele J (2016) Histidine phosphorylation relieves copper inhibition in the mammalian potassium channel KCa3.1. elife 5:pii:e16093
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kalagiri, R., Adam, K., Hunter, T. (2020). Empirical Evidence of Cellular Histidine Phosphorylation by Immunoblotting Using pHis mAbs. In: Eyers, C. (eds) Histidine Phosphorylation. Methods in Molecular Biology, vol 2077. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9884-5_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9884-5_12
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9883-8
Online ISBN: 978-1-4939-9884-5
eBook Packages: Springer Protocols