Assays for tyrosine phosphorylation in human cells

Monica Kruk, Naomi Widstrom, Sampreeti Jena, Nicole L. Wolter, John F. Blankenhorn, Ibrahim Abdalla, Tzu Yi Yang, Laurie L. Parker

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Tyrosine kinases are important for many cellular processes and disruption of their regulation is a factor in diseases like cancer, therefore they are a major target of anticancer drugs. There are many ways to measure tyrosine kinase activity in cells by monitoring endogenous substrate phosphorylation, or by using peptide substrates and incubating them with cell lysates containing active kinases. However, most of these strategies rely on antibodies and/or are limited in how accurately they model the intracellular environment. In cases in which activity needs to be measured in cells, but endogenous substrates are not known and/or suitable phosphospecific antibodies are not available, cell-deliverable peptide substrates can be an alternative and can provide information on activation and inhibition of kinases in intact, live cells. In this chapter, we review this methodology and provide a protocol for measuring Abl kinase activity in human cells using enzyme-linked immunosorbent assay (ELISA) with a generic antiphosphotyrosine antibody for detection.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
EditorsBenjamin A. Garcia
PublisherAcademic Press Inc.
Pages375-406
Number of pages32
ISBN (Print)9780128186695
DOIs
StatePublished - 2019

Publication series

NameMethods in Enzymology
Volume626
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Abl kinase
  • Cell penetrating peptide
  • K562 cells
  • Kinase substrate
  • Tyrosine kinase assay

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural

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