![]() These imagers are quite pricey, however – many departments will purchase one for all of their investigators to use. It’s also nice to have your image in digital form already, instead of having to scan in a physical blot once you’ve developed it. The imager then automates the process of developing the blot: just hit the “image” button, and it will take multiple different exposures of the blot, and save all the images so you can compare them and choose your favorite.ĬCD imagers are a lot more convenient then using x-ray film, and they circumvent the problem of handling film and having to use a special dark room for developing. The only difference is that once you’ve taken your membrane out of the developing solution, you can place it directly on the imaging stage of the CCD imager. To prepare your blot for a CCD imager, you follow basically the same procedure as you would use when prepping for x-ray film. These machines look a lot like the imager you would use to image a DNA gel – and in fact, many imagers can handle both kinds of detection. These imagers contain a detector that translates the chemical signal (resulting from the HRP-ECL reaction described above) to a digital image. Using a CCD imager is the more contemporary, hands-off way to image a western blot. I’ve seen these around, but I’ve never actually seen anyone use them!ĭeveloping x-ray film can be slow and a little messy, but it gives you a lot of control over the developing process, including time of exposure and even the ability to expose your blot to the same piece of film multiple times. ![]() You can then pop the film directly into the film developer – or, if your lab is really old school, you can develop the film by hand using successive baths of developing solutions. Open the cassette carefully to avoid sliding the blot and film relative to each other (which will result in blurry bands). Every protein-antibody combination has a different optimal exposure time, but you’ll usually want to expose the film for 1-10 minutes. Place your blot face down on a piece of film, and close the developing cassette – this will press the blot to the film and eliminate any other possible source of light, making sure you get a clear, strong signal. The developing room should have a red light, which is okay to keep on while handling film. Then, seal it up in plastic wrap to keep it from dripping, and trot over to the developing room. To develop your blot, you simply soak the surface of the blot in developing solution for 1-2 minutes. You must be absolutely sure to handle the film in a sealed dark room, so as not to expose the film to bright ambient light (it’s a rite of passage for new researchers to expose an entire box of expensive film to light through poor handling – the shame you experience will keep you from ever doing it again!). ![]() In the case of developing a blot, the light comes from the chemical reaction between horseradish peroxidase (HRP), which is conjugated to your secondary antibody, and the ECL solution you use to detect the signal. When you take a photo, the image is imprinted on the film due to varying amounts of ambient light that reach the film surface, which is why photos taken on a bright sunny day look blown out, while photos taken in the dark are almost black. The x-ray film will act just like photographic film, which “bleaches” when exposed to light. Read on for more info about different imaging modalities… X-ray filmĭeveloping your blot on x-ray film is the traditional way to detect protein signals. The method you choose will largely depend on the type of equipment that’s available in your lab, and will also affect the reagents you use to detect your protein signals. The last step in western blotting is imaging the blot – this is the moment of truth, when you finally get to see the results of the experiment you’ve been working on for so long! There are a variety of different ways to image your blot.
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