Hello,
I have been doing FRAP on GFP tagged proteins using CLSM510META without any problem, but recently tried bleaching DsRed (monomer) tagged proteins. I used HeNe laser (543 nm) up to 100% power to bleach but I do not see any bleaching (~100 iterations). I also tried 488nm laser along with 543nm laser, without any bleaching..!
I would appreciate, if you have any suggestion on this.
Thank you.
Hello Ashis, DsRed monomeric has a very high photobleaching speed (0.3-0.4 ms when excited at 532 nm at high power (50-100mW)). But the problem arise when DsRed polimerizes. This can happen when DsRed is highly expresed and polimerization implies an increase in photostability. That means that sometimes you kill your cells before DsRed protein is photobleached. In other hand, what is the HeNe laser power? And its age? Regards, Alberto
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