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I was hoping to see more from others more learned than I before I open my mouth on this one but it seems they will be silent.  As far as I can tell, the best argument for the strip bleaching paradigm is that it allows you to hit a broad, ideally homogenous, zone of the nucleus and apply boundary conditions to the zones where protein travels from and to.  These boundary conditions allow one to get to association and dissociation constants and thus residence times.  G. Carrero and M. Hendzel do just this in their 2003 Methods paper (Methods, 29 (2003) 14-28).  Warning for the math disinclined, this paper is seriously math heavy.

Where I think one might run into problems is if the zones are not, in fact, homogenous.  If one has an area with altered affinity, it winds up contributing to variability in recovery time since it is likely to be placed randomly with regard to the bleach strip.  The way to get around this, assuming one can see this area, is to spot bleach to a fixed radius around the zone of altered affinity.  This results in better reproducibility but I have yet to see a work get to the point of calculating association/dissociation constants from a point bleach.  One problem is that one never bleaches just the zone of altered affinity and thus are susceptible to alterations in the levels of non zone fluorophore in the bleach spot.

Other sources of variability in these experiments abound.  The health of the cells is CRITICAL.  Any source of stress can alter HSPs and seriously start to alter recovery times.  Temperature is likewise critical.  Protein expression levels must be kept within reasonable limits both to promote cellular health and to avoid aggregation.  Take heart, you are not the only one to run into such problems.

Hope this helps and please keep in mind that I am NOT a FRAP guru, at least not yet,

D. Scott Snyder
Lab Manager, Integrated Microscopy Core
Baylor College of Medicine 


 

Hello everyone,
 
I've been doing FRAP experiments as off late and I'm running into some problems. There seem to be two formula's used for curvefitting.  One is based on strip bleaching and the other one on spot bleaching but seems to be aplicable for bigger regions. Can somebody explain me the difference and tell me which one will give better results.
 
Another question is coming from this I seem to have a high variance in my mobile/immobile fractions, I wondered if this is a common problem or if it is sample dependant.
 
The last question I have is based on calculating residence times. Mostly people caluclate the amount of time a protein stays in a certain region, does anybody know a possibility to calculate the amount of time a protein is bound to DNA, or other non moving structures inside a cell.
 
Looking forward to the answers
 
Joost Willemse Msc.
Lab. of Molecular Biology
Wageningen UR
+31(317)484574