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A Gathered Report on Fluorescent Recovery After Photobleaching

Discussion in 'DAT Discussions' started by creative8401, Jun 5, 2008.

  1. creative8401

    creative8401 Im Anush Hayastan

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    Hi everyone. I did this little research of mine on FRAP. I have no idea what type of question they ask on the exam, nor am I interested in knowing in advance, but I’ve gathered this report since for me, finding relevant information on this technique is cumbersome. As far as I know, you get one or two questions dealing with this technique (or none at all), and is it worth it to know? That is up to you. I’d like to thank TheWiredNerve and all the students who contributed to the thread: http://forums.studentdoctor.net/showthread.php?t=442518, I’ve gathered their data and a number of information from other sources. Let’s get started.

    FRAP, which stands for fluorescent recovery after photobleaching, is a technique that monitors movement within say a cell membrane. The area of interest is tagged with fluorescent markers, and bleached afterwards. What this means is that within a given area, the fluorescent markers are removed. Let’s take a look:


    http://www.olympusfluoview.com/applications/flipandfrap.html

    In A, the area in pink is used to represent the fluorescent markers. The square is B represents the bleached area, meaning that the fluorescent markers that were present in A were removed. After some time, the fluorescent markers from the surrounding areas moved within the bleached area. What does it show? It means that the membrane, or what is shown above (maybe a protein) has fluidity of some sort and allows for the movement of fluorescent markers into the bleached area.

    Experimental Setup
    “First thing that is done in FRAP is to tag the specific protein you are examining with a fluorescent compound, usually green fluorescent proteins (GFP) or an antibody of some sort. These fluorescent groups are attached to the proteins using various genetic recombinant techniques such as DNA cloning. A portion of the membrane is then exposed to a very strong laser beam. This laser essentially bleaches the proteins of the fluorescent tags so that the area of the membrane treated with the laser is no longer viewed as green under a confocal microscope. The rate at which tagged proteins move into the now bleached area is called the "fluorescence recovery." – 8/31/07 thanks to TheWiredNerve

    Let’s take a look at this very helpful animation: (if you get an ad, press “skid this ad”): http://www.dnatube.com/view_video2.php?viewkey=125ed6c3f678819b6e05

    “So the gist of it is that if the membrane has a fluid quality to it, the fluorescence will recover, if not, then you will see this black patch of area indicating that no movement is taking place. Ok…seems nothing complicated. I’m thinking that its like a mini-GPS system, where it tracks and monitors movements within the cell membrane. “

    Besides using this technique on lipid composition, it is also used to tag proteins. Since proteins can be both integral and peripheral (in regards to cell membrane), it can be used to again measure their movement. The “TheWiredNerve” brought up a good point – lipid rafts.


    http://www.bms.ed.ac.uk/research/others/smaciver/Cyto-Topics/lipid_rafts_and_the_cytoskeleton.htm

    As shown above (link), rafts are regions where the phospholipids fatty acid tails are straight, meaning that the bonds are saturated. Remember that unsaturated (presence of double bonds) leads to cis conformation, and hence bending of the tails. Saturaded (single carbon to hydrogen) bonds all for close packing of the area. If a protein is present in such an area, then it will not be able to move. Imagine taking a button and sewing in a tight network of threads onto a shirt – its movement in any aspect is impossible. (I think of these analogies to help us understand, they should not be taken word for word, but to develop the underlying concept)

    Let’s make this a bit complicated: Graphs

    http://www.bio.davidson.edu/Courses/Molbio/FRAPx/FRAP.html

    A graphical analyses is possible of the technique. This is my understanding:

    1) Point 1 represents the initial area that is fluorescencing (I doubt such a word exists, lol). It basically represents the initial area that is “lighting” up.

    2) Point 2 represents the drop is fluorescence due to photobleaching. A bunch of fancy words meaning the area that was cleared up. Imagine you get a stain on your shirt after grabbing a snack at 2AM. You grab one of those Clorox bleach pens (the ones you see on commercials), and clear the area. That clear area is point 2. Why doesn’t it reach zero? This is probably due to the technique in which the laser beam cannot bleach the entire area so you are still left with some fluorescent markers (this is why in the animation, there were some fluorescent markers left).

    3) If the area is fluid, then the neighboring fluorescent markers start to diffuse into the area

    4) This is used to represent equilibrium. It never reached point 1 because you have a lesser amount of fluorescence markers.

    The notes I’ve gathered are my understanding of the technique. I neither have a Ph.D. or am a professor of this stuff, and if some concepts are not addressed or clearly represented, it would be great to add more.
     
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  3. happyasaclam88

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    this is an incredibly clear and friendly explanation. thank you so much!
     
  4. nemosundori

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    is this FRAP test still around? Anyone had it recently?...
     
  5. Sea of ASH

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    this was used to show the membrane fluidity. this test can be used to expiriment theories that are related to the membrane, from my understanding.

    wikipedia has some good stuff on it: http://en.wikipedia.org/wiki/Fluorescence_recovery_after_photobleaching#Applications_Outside_the_Membrane

    Applications Outside the Membrane
    FRAP can also be used to monitor proteins outside the membrane. After the protein of interest is made fluorescent, generally by expression as a GFP fusion protein, a confocal microscope is used to photobleach and monitor a region of the cytoplasm, mitotic spindle, nucleus, or another cellular structure. The mean fluorescence in the region can then be plotted versus time since the photobleaching, and the resulting curve can yield kinetic coefficients for the protein's binding reactions and/or the protein's diffusion coefficient in the medium where it is being monitored. The analysis is most simple when the curve is dominated by only the diffusional or only the binding components. For a circular bleach spot and diffusion-dominated recovery, the fluorescence is described by the Soumpasis equation and involves modified Bessel functions:
    f(t)=e-h(I0(h)+I1(h)) where h=r2/(2*Df*t); r=radius of bleach spot; t=time; Df=diffusion coefficient; f(t) is the normalized fluorescence (goes to 1 as t goes to infinity).
    For a binding-dominated reaction, in which the diffusion is much faster than the unbinding of the bleached protein and subsequent binding of unbleached protein, it is given by
    f(t)=1-Beqe-kofft where Beq is the fraction of the protein that is bound to other structures in the photobleached region at equilibrium, and koff is the dissociation constant for the binding. Sometimes there are multiple binding states in which case there are just more exponential terms of the same form. Many FRAP recoveries are not dominated overwhelmingly by just diffusion or just binding, so their curves are more complex; FRAP recoveries are analyzed in much more detail in Sprague and Pego et al. (wikipedia.com)
     
    #4 Sea of ASH, Jun 6, 2008
    Last edited: Jun 6, 2008
  6. smile101

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    I dunno if I am going to have the question on FRAP again (I had it when I took DAT the first time), but thanks for sharing ur research. True passion!!!
     

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