Oh FRAP!

One of my favorite experiments to do in my lab involves shooting cells with lasers. Yeah, that’s right…shooting cells with lasers! It’s part of a really cool procedure called fluorescence recovery after photobleaching or FRAP for short.

The fluorescent proteins that I wrote about before are useful way beyond just making glowing fish. They can also be fused to other proteins. This is done at the DNA level by taking the gene for one protein and fusing it to the gene for a fluorescent protein. The molecular machinery inside the cell transcribes this DNA onto a single RNA which is then translated into a single protein called a fusion protein. Fusion proteins can often do the same thing that the original protein did. (It’s very important to test that this is the case. Sometimes tacking on an extra piece can disrupt the protein’s function!) Here’s a picture of a fusion protein that I made. The protein is in the nucleus of a living cell, as seen under a fluorescence microscope. I have done various tests to make sure that it still does what the normal protein does. So far, it has passed all of my tests in cultured cells (in a Petri dish, basically), and I am now working to do similar tests in living flies.

So, what is FRAP? FRAP is a technique that can be used to study how fast fluorescently tagged proteins move around inside cells. The idea is actually pretty simple: You take a laser and zap the fluorescently-labeled protein in a little spot. (You do all of this with a high-tech microscope.) The laser is so intense that the fluorescent label goes dark or photobleaches. This is sort of like what happens when you leave colored construction paper out in the light for a long time. The dye molecules in the paper undergo photochemical reactions (chemical reactions caused by light) that cause them to lose their color. Similarly, a fluorescent molecule photobleaches when it undergoes a photochemical reaction that turns it permanently into something non-fluorescent.

The rest of the fluorescently-labeled protein, however, is still okay. It still glows as before. If the protein is free to move around, then the bleached protein inside the spot will diffuse out and the fluorescent protein surrounding the spot will diffuse in. You can determine how mobile a fluorescent fusion protein is by measuring how long it takes for this exchange to occur. The result is a recovery curve, like the one below. Proteins that are free-floating in the cell recovery quickly after photobleaching. Proteins that are stuck to larger things recover more slowly. You can figure out a lot about what a protein is doing in a cell by comparing FRAP curves of different mutants under different conditions.

In addition to FRAP, there are even more advanced fluorescence techniques that you can use to study how proteins behave in living cells…more on those later.