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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