Back to article: Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways

FIGURE 16: iFRAP analysis of Replication protein A and kinetochore subunit Mtw1. (A) Principle of iFRAP method. An image is acquired immediately before (green data point) and after (red data point) laser bleaching of one of two subnuclear foci (indicated by dashed red circle). The fluorescence intensity of the focus before bleaching is set to 1 and all subsequent fluorescence measurements are normalized accordingly. Fstart indicates the fluorescence intensity of the bleached focus immediately after laser bleaching. Fend indicates the plateau fluorescence intensity that the bleached focus approaches during redistribution. Fmax indicated the maximum fluorescence intensity that the bleached focus can reach at the end of the experiment assuming 100% redistribution. The mobile protein fraction can be calculated as (Fend − Fstart)/(Fmax − Fstart). (B and C) Cells with two foci were subjected to photobleaching of one focus (indicated by dashed red circle) at t = 8s. Fluorescence redistribution was quantified at subsequent time points. The redistribution half-time (T1/2) is indicated with 95% confidence intervals in parentheses. Scale bar, 3 µm. The protein mobile fraction is indicated ± standard deviation. (B) RPA dynamically exchanges at DNA damage-induced foci. Cells expressing Rfa1-YFP (ML306) were grown in the presence of 200 µg/ml Zeocin for 2 hours to induce DNA repair foci. (C) Mtw1 is a stable component of the kinetochore. Cells expressing Mtw1-GFP [368] were arrested in M phase by treatment with 10 µg/ml nocodazole for 2 hours. One-phase association non-linear regression was performed using GraphPad Prism software. Error bars indicate SEM.

368. Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS, O'Shea EK (2003). Global analysis of protein localization in budding yeast. Nature 13(4): 367–374. 425(6959): 686-691. doi: 10.1038/nature02026

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