Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocalHello all,
@Matt: you wrote that you study protein-turnover. FRAP is a technique for
protein dynamics, to my understanding turnover would mean the new
synthesis of a protein and the degradation processes. But I guess you meant
dynamics.
For the analysis the follwing link might be useful.
http://www.embl.de/eamnet/frap/FRAP6.htmlTo answer your questions directly:
"Firstly how is it best to determine the parameters for FRAP, i.e. how do you
know how many prebleach, bleach and post bleach images to capture? "
Usually I did not more than 10 prebleach images. This should just show you a
horizontal line on your graph, as a starting point. For the bleaching I used also
488nm when bleaching GFP, but I also found 405nm works nicely (and usually
you bleach so fast, that I think photodamage is not an issue). For the
important images, the postbleach, just try how long you need to reach
the "plateau", and add a bit to be sure to get all data.
"On previous attempts I have used 4 bleach iterations using the fly mode. I
assume that you should be able to see in the viewer as the ROI bleaches?
does the size of the ROI have a large impact on how long the area takes to
bleach?"
Yes and yes.
"So far I have not been able to bleach the region using our argon laser 488 line
at 100% power. Should I use more than one laser line?"
Thats strange. If you have a 405 line, try it once. Maybe you should check
the power output of your Argon laser.
"I just find this odd as i'd expect the region to bleach quickly at 100% power
output."
I totally agree.
Best regards and good luck.
Florian
Locked
