Proximity ligation assay to detect and localize the interactions of erα with PI3-k and src in breast cancer cells and tumor samples

Abstract

In situ proximity ligation assay (PLA) is a powerful method for detection, localization, and quantifi cation of proteins, protein–protein interactions, and posttranslational modifi cations. Proteins detected by two specifi c antibodies are recognized by proximity probes conjugated with complementary oligonucleotides to allow the formation of circular DNA probes when bound in close proximity. Subsequent amplifi cation of this DNA can then be visualized. Here, we describe the in situ PLA method for the detection of the ERα/Src/PI3K complex in breast cancer. We used two different techniques for detecting the signals: fl uorescent detection for cell line analysis and bright-fi eld revelation, which is better suited to clinical analysis of patient samples.