Whitley Vistisen posted an update 4 months, 1 week ago
The fluorescent probes were detected by immunoblotting using a monoclonal antibody specific to GFP that recognizes YFP and CFP. CFP was excited at nm, and emission was detected between and nm.YFP was excited at nm, and emission was detected from to nm.The images were collected using a oil objective, and recorded in bit mode at a scan rate of. FRET was measured based on the increase in the CFP signal following photobleaching of the YFP.Membrane targeting of fluorescent proteins by the L, S, and LAT sequences.LAT is the first amino acids of LAT and contains the extracellular and transmembrane domains of fulllength protein, plus the membraneproximal cysteines that are palmitoylated.The sites of myristoylation for each of the constructs are indicated.Fraction represents the top of the gradient, and the raft and TXS membrane fractions occurred in the indicated gradient fractions.Molecular weights are in the thousands, and the relative amount of each protein associated with the raft fraction is indicated.Four images were acquired for each region of interest: an image of YFP fluorescence before bleach an image of YFP fluorescence after bleach. In addition, to correct for bleaching of CFP during YFP bleaching, cells expressing CFP alone were taken through all the steps of FRET measurement.Cells expressing only YFP were used to determine the YFP bleedthrough into the CFP channel. LAT contains the transmembrane domain of LAT but lacks its cytoplasmic domain with associated linker functions because of palmitoylation of a set of membraneproximal cysteine residues. Detection of FRET by acceptor photobleaching.LynCFPYFP is membraneanchored protein and the CFP and YFP are separated by only two amino acids.The CFPTDNYFP is a soluble protein that contains a amino acid linker between the CFP and the YFP.For each, FRET was detected by measuring for an increase in CFP fluorescence following photobleaching of the YFP.The images were collected in the CFP photobleaching of the acceptor.The yellow square in each image marks the region that was photobleached in the YFP channel, and an enlarged view of the region is shown in the inset.Accompanying the images are plots of the membrane fluorescence intensity in each channel before photobleaching.In the graphs, theyaxis represents relative fluorescence intensity of the plasma membrane, and thexaxis corresponds to the distance around the periphery of the cell.The vertical lines indicate the boundaries of the region that was photobleached.If random, then E increases with acceptor concentration.E was determined over a range of acceptor intensities, and the values are plotted versus the prebleach acceptor intensity.In D, the donoracceptor ratio was: for each cell measured.Furthermore, to show that observed changes in CFP fluorescence following YFP photobleaching were specific to FRET, measurements were made using cells expressing a second control peptide Meropenem containing CFP and YFP separated by a amino acid linker. Accompanying the images are plots of the fluorescence intensity values of the outer membrane in the CFP and YFP channels before photobleaching.The vertical lines in the plots mark the boundaries of the bleached regions and show the photobleaching extinguished of the YFP fluorescence.In contrast, photobleaching YFP of the CFPTDNYFP resulted in no detectable change in its CFP fluorescence.