Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines.

Mark Aspinall-O'Dea, Andrew Pierce, Francesca Pellicano, Andrew Williamson, Mary T Scott, Michael J Walker, Tessa L Holyoake, Anthony Whetton

Research output: Contribution to journalArticlepeer-review

Abstract

This protocol describes a highly reproducible antibody-based method that provides protein level and phosphorylation status information from nanogram quantities of protein cell lysate. Nanocapillary isoelectric focusing (cIEF) combines with UV-activated linking chemistry to detect changes in phosphorylation status. As an example application, we describe how to detect changes in response to tyrosine kinase inhibitors (TKIs) in the phosphorylation status of the adaptor protein CrkL, a major substrate of the oncogenic tyrosine kinase BCR-ABL in chronic myeloid leukemia (CML), using highly enriched CML stem cells and mature cell populations in vitro. This protocol provides a 2.5 pg/nl limit of protein detection (
Original languageEnglish
Pages (from-to)149–168
JournalNature protocols
Volume10
Issue number1
DOIs
Publication statusPublished - 18 Dec 2014

Research Beacons, Institutes and Platforms

  • Manchester Cancer Research Centre

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