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

doi:10.1038/nprot.2015.007

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

Division of Cancer Sciences (L5)

University of Glasgow

Research output: Contribution to journal › Article › peer-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 language	English
Pages (from-to)	149–168
Journal	Nature protocols
Volume	10
Issue number	1
DOIs	https://doi.org/10.1038/nprot.2015.007
Publication status	Published - 18 Dec 2014

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Manchester Cancer Research Centre

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10.1038/nprot.2015.007

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Aspinall-O'Dea, M., Pierce, A., Pellicano, F., Williamson, A., Scott, M. T., Walker, M. J., Holyoake, T. L., & Whetton, A. (2014). Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines. Nature protocols, 10(1), 149–168. https://doi.org/10.1038/nprot.2015.007

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

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 (",

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Aspinall-O'Dea, M, Pierce, A, Pellicano, F, Williamson, A, Scott, MT, Walker, MJ, Holyoake, TL & Whetton, A 2014, 'Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines.', Nature protocols, vol. 10, no. 1, pp. 149–168. https://doi.org/10.1038/nprot.2015.007

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AU - Aspinall-O'Dea, Mark

AU - Pierce, Andrew

AU - Pellicano, Francesca

AU - Williamson, Andrew

AU - Scott, Mary T

AU - Walker, Michael J

AU - Holyoake, Tessa L

AU - Whetton, Anthony

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

Abstract

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