yet another FRET question
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Romin, Yevgeniy/Sloan Kettering Institute |
yet another FRET question
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello everybody Lately I've been getting mixed responses to the following question - How much can one trust FRET measurements in the cell membrane? There are many papers published showing FRET in the membrane (proteins involved in cell movement, actin polymerization, etc.) But I've also heard statements made that because of the dynamics in the cell membrane there is great possibility of random FRET occurring and the FRET data acquired from cell membranes is quite often artifactual. Has anybody had any experience with this issue before? Thanks in advance for your always helpful responses, --------------------------------------------------- Yevgeniy Romin Digital Microscopist Memorial Sloan-Kettering Cancer Center Molecular Cytology Core Facility 1275 York Ave. Box 333 New York, NY 10065 Tel.646-888-2186 Fax. 646-422-0640 --------------------------------------------------- ===================================================================== Please note that this e-mail and any files transmitted with it may be privileged, confidential, and protected from disclosure under applicable law. If the reader of this message is not the intended recipient, or an employee or agent responsible for delivering this message to the intended recipient, you are hereby notified that any reading, dissemination, distribution, copying, or other use of this communication or any of its attachments is strictly prohibited. If you have received this communication in error, please notify the sender immediately by replying to this message and deleting this message, any attachments, and all copies and backups from your computer. |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Yevgeniy, Because membrane is confined space, the local concentration (if you can define concentration in 2D space) of your FRET probe tends to be higher than would be in other organelles, and this could lead to artifactual "reabsorption" of the donor's fluorescence. You will not get "random FRET" because, at conceivable concentration of the probe, the average distance between the donor and acceptor would not be shorter than Foerster distance when they are not interacting directly. If you rely on spectroscopic detection, it is hard to distinguish FRET signal from the reabsorption. My only advice is to keep the concentration of your FRET probe as low as possible. You can probably measure the critical concentration by using non-FRET control with various concentrations. If you have access to one of those FLIM systems, measurement of donor life time will give you a definite answer. Ippei Kotera Tanz CRND Tanz Neuroscience Building, Room 221 University of Toronto 6 Queen's Park Crescent West Toronto, Ontario M5S 3H2 Phone: (416)978-2503 On 26/01/2011 11:01 AM, > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello everybody > > Lately I've been getting mixed responses to the following question - How much can one trust FRET measurements in the cell membrane? There are many papers published showing FRET in the membrane (proteins involved in cell movement, actin polymerization, etc.) But I've also heard statements made that because of the dynamics in the cell membrane there is great possibility of random FRET occurring and the FRET data acquired from cell membranes is quite often artifactual. Has anybody had any experience with this issue before? > > Thanks in advance for your always helpful responses, > > --------------------------------------------------- > Yevgeniy Romin > > Digital Microscopist > Memorial Sloan-Kettering Cancer Center > Molecular Cytology Core Facility > 1275 York Ave. Box 333 > New York, NY 10065 > Tel.646-888-2186 > Fax. 646-422-0640 > --------------------------------------------------- > > > > ===================================================================== > > Please note that this e-mail and any files transmitted with it may be > privileged, confidential, and protected from disclosure under > applicable law. If the reader of this message is not the intended > recipient, or an employee or agent responsible for delivering this > message to the intended recipient, you are hereby notified that any > reading, dissemination, distribution, copying, or other use of this > communication or any of its attachments is strictly prohibited. If > you have received this communication in error, please notify the > sender immediately by replying to this message and deleting this > message, any attachments, and all copies and backups from your > computer. > |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Yevgniy and Ippei, I've been trying to measure Acceptor Photobleaching FRET on membrane proteins for a while, using confocal microscopy. When the expression of my tagged-receptor is high enough that it can be comfortably detected by my confocal system, I get a background FRET of about 8%. The way I tested this is by co-expressing two proteins that should not interact (as far as we know). When I get FRET of about 20%-30% I am confident it is true FRET. The problem though is that the variability among individual cells is quite high, so large number of measurements need to be done. I tried to reduce the expression to see if I can reduce "random FRET" but then I have the problem that the intensity is too low for my confocal and fluorophores system. Regards, Pablo -- Pablo German PhD Candidate Plant and Food Research Private Bag 92169 Auckland Mail Centre Auckland 1142 New Zealand DDI: (09) 925-7107 Mobile: 0210459406 On 27 January 2011 06:13, Ippei Kotera <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi Yevgeniy, > > Because membrane is confined space, the local concentration (if you can > define concentration in 2D space) of your FRET probe tends to be higher > than would be in other organelles, and this could lead to artifactual > "reabsorption" of the donor's fluorescence. You will not get "random > FRET" because, at conceivable concentration of the probe, the average > distance between the donor and acceptor would not be shorter than > Foerster distance when they are not interacting directly. > > If you rely on spectroscopic detection, it is hard to distinguish FRET > signal from the reabsorption. My only advice is to keep the > concentration of your FRET probe as low as possible. You can probably > measure the critical concentration by using non-FRET control with > various concentrations. If you have access to one of those FLIM systems, > measurement of donor life time will give you a definite answer. > > > Ippei Kotera > > Tanz CRND > Tanz Neuroscience Building, Room 221 > University of Toronto > 6 Queen's Park Crescent West > Toronto, Ontario M5S 3H2 > Phone: (416)978-2503 > > > On 26/01/2011 11:01 AM, >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Hello everybody >> >> Lately I've been getting mixed responses to the following question - How much can one trust FRET measurements in the cell membrane? There are many papers published showing FRET in the membrane (proteins involved in cell movement, actin polymerization, etc.) But I've also heard statements made that because of the dynamics in the cell membrane there is great possibility of random FRET occurring and the FRET data acquired from cell membranes is quite often artifactual. Has anybody had any experience with this issue before? >> >> Thanks in advance for your always helpful responses, >> >> --------------------------------------------------- >> Yevgeniy Romin >> >> Digital Microscopist >> Memorial Sloan-Kettering Cancer Center >> Molecular Cytology Core Facility >> 1275 York Ave. Box 333 >> New York, NY 10065 >> Tel.646-888-2186 >> Fax. 646-422-0640 >> --------------------------------------------------- >> >> >> >> ===================================================================== >> >> Please note that this e-mail and any files transmitted with it may be >> privileged, confidential, and protected from disclosure under >> applicable law. If the reader of this message is not the intended >> recipient, or an employee or agent responsible for delivering this >> message to the intended recipient, you are hereby notified that any >> reading, dissemination, distribution, copying, or other use of this >> communication or any of its attachments is strictly prohibited. If >> you have received this communication in error, please notify the >> sender immediately by replying to this message and deleting this >> message, any attachments, and all copies and backups from your >> computer. >> > |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Pablo, Acceptor photobleching alone cannot distinguish true FRET from reabsorption of fluorescence, so it's a good idea to have non-interacting FPs as a negative control. Is the false FRET efficiency with those control FPs stable around 8% among different cells? If so, then variation in donor/acceptor interaction might be the reason, not the measurement. Alternatively, you can try donor photobleaching to estimate the true FRET efficiency. The donor photobleaching rate is not affected by reabsorption, so it might be more appropriate for membrane proteins. Good luck, Ippei Kotera Tanz CRND Tanz Neuroscience Building, Room 221 University of Toronto 6 Queen's Park Crescent West Toronto, Ontario M5S 3H2 Phone: (416)978-2503 |
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