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Empirical Evidence of Cellular Histidine Phosphorylation by Immunoblotting Using pHis mAbs

  • Rajasree Kalagiri
  • Kevin Adam
  • Tony HunterEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2077)

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.

Key words

Posttranslational modification Histidine phosphorylation pHis antibodies Western blotting Immunoblotting Phosphoramidate bond 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Molecular and Cell Biology LaboratorySalk Institute for Biological StudiesLa JollaUSA

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