Detect clustering in cells using Homo-FRET

Abstract

In this research homo-FRET was used to detect clustering of EGFR in cells. To test this method reference constructs were made containing (2x)FKBP. A wide field microscope, polarizing beamsplitter and two cameras where used to detect polarizations. As a labeling fluorophore mCherry was used instead of the common mGFP because the autofluorescence is believed to be lower in the red spectrum. The best result obtained so far is a drop of 7.8 ± 6.8% in the case of FKBP and 11.3 ± 6.1% in the case of 2xFKBP upon adding AP. EGFR samples where also stimulated with EGF but a significant drop was not observed yet. The main problem encountered was registrating the two images.