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Fig. 3. FRET analysis shows the direct interaction of PGI/AMF and FN at pH 5 but not at pH 7.5. NIH-3T3 cells incubated with FN-488 for 30 minutes at pH 7.5 in complete HEPES-buffered medium were then incubated with PGI/AMF-568 for 30 minutes at 37°C at either pH 7.5 in complete HEPES-buffered medium (A-D) or at pH 5 in complete MES-buffered medium (E-H), washed with PBS/CM, fixed with 3% paraformaldehyde and the cover slips mounted in Airvol for confocal photobleaching FRET analysis. Single scan images before bleaching of the acceptor PGI/AMF-568 (A,E) and donor FN-488 (C,G) were collected and then the acceptor was bleached to 60% to 90% in the zone of interest (indicated by the box). New single scan images were then acquired simultaneously for both the donor (B,F) and the acceptor (D,H). Donor images are presented in pseudocolor (see bar to right) to highlight increased donor fluorescence after acceptor photobleaching. The extent of bleaching (% bleach) and increased donor fluorescence (% FRET) in the bleached region was quantified and is presented in table form (I) for FN-488 and PGI/AMF-568 as donor/acceptor pairs at both pH 5 and 7.5. Negative controls include donor alone or acceptor alone at pH 7.5 or 5.0 and as a positive control FRET between FN-488 and FN-568 was measured, as indicated. P values were determined by ANOVA relative to the FN-488 and PGI/AMF-568 pair at pH 7.5.
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