Open Access Open Badges Research article

Tyrosine phosphorylation profiling via in situ proximity ligation assay

Lioudmila Elfineh1, Christina Classon1, Anna Asplund1, Ulf Pettersson1, Masood Kamali-Moghaddam1 and Sara Bergström Lind12*

Author Affiliations

1 Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden

2 Department of Chemistry-BMC, Analytical Chemistry, Science for Life Laboratory, Uppsala University, Box 599, SE-751 24 Uppsala, Sweden

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BMC Cancer 2014, 14:435  doi:10.1186/1471-2407-14-435

Published: 13 June 2014



Tyrosine phosphorylation (pTyr) is an important cancer relevant posttranslational modification since it regulates protein activity and cellular localization. By controlling cell growth and differentiation it plays an important role in tumor development. This paper describes a novel approach for detection and visualization of a panel of pTyr proteins in tumors using in situ proximity ligation assay.


K562 leukemia cells were treated with tyrosine kinase and/or phosphatase inhibitors to induce differences in pTyr levels and mimic cells with different malignant properties. Cells were then probed with one antibody against the pTyr modification and another probe against the detected protein, resulting in a detectable fluorescent signal once the probes were in proximity.


Total and protein specific pTyr levels on ABL, SHC, ERK2 and PI3K proteins were detected and samples of control and treated cells were distinguished at the pTyr level using this novel approach. Promising results were also detected for formalin fixed and paraffin embedded cells in the micro array format.


This application of in situ proximity ligation assay is valuable in order to study the pTyr modification of a panel of proteins in large data sets to validate mass spectrometric data and to be combined with tissue microarrays. The approach offers new opportunities to reveal the pTyr signatures in cells of different malignant properties that can be used as biomarker of disease in the future.

Cancer biomarkers; Protein signaling; Protein tyrosine phosphorylation; in situ proximity ligation assay (in situ PLA)