Colocalisation Assay The effect of point mutations on the pro-apoptotic protein BAX were examined using HCA. CHO cells were transfected with various GFP-tagged Bax mutants. The cells were then stained with a mitochondrial marker (red stain) and the co-localisation of GFP to the mitochondria (colocalisation of green to red stain) quantified (right panel). In this example the left picture shows BAX wild type while the middle picture is the co-localisation positive control (YFP with mitochondrial targeting sequence).

Colocalisation Assay

The encoding of cell signal tranduction is not just dependent on the phosphorylation state of the signalling proteins. Another important factor is the position of signalling proteins in relation to other cellular components. The power of HCA is that it gives detailed information on where a particular component is located in the cell. Furthermore, if an experiment probes for multiple cellular proteins, then the relationship of these proteins to one another can be fully appreciated using HCA algorithms that measure the co-localisation of the stain. This is far superior to flow cytometric-type assays and is why microscopy-based HCA is the only true method for doing High Content Screening.

Courtesy of Andrew Gillmore, The University of Manchester, UK