FLIP analysis of mCherry::ubiquitin and Q82::GFP. Worms co-expressing an mCherry::ubiquitin fusion protein with Q82::GFP were subjected to FLIP analysis of the mCherry::ubiquitin protein to study mobility of ubiquitin. Fluorescence intensity is indicated by a heat map of mCherry::ubiquitin prior to bleaching and at various times after commencement of repeated bleach pulses. Red squares indicate regions where bleach pulses were directed, black and white squares indicate regions that were quantitatively analyzed for fluorescence loss, and yellow squares indicate regions in non-bleached cells that were used to control for acquisition photobleaching. Separate experiments were carried out in which bleaching was directed to the cytoplasm (A) or the aggregate of Q82::GFP (B). A quantitative analysis (C) was carried out to analyze fluorescence loss over time. Results indicate no loss of mCherry fluorescence in aggregates when bleaching was directed to either a separate region within the aggregate itself (blue diamonds) or an area in the cytoplasm. The loss of fluorescence in the cytoplasm when a region within the cytoplasm was bleached indicates the effectiveness of the bleaching protocol (red squares), while the limited loss of fluorescence in the cytoplasm when a region within the aggregate was bleached indicates the limited access of mCherry::ubiquitin to the Q82::GFP aggregates.
Skibinski and Boyd BMC Cell Biology 2012 13:10 doi:10.1186/1471-2121-13-10