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. 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. Read on for more info about different imaging modalities…
Developing your blot on x-ray film is the traditional way to detect protein signals. The x-ray film will act just like photographic film, which “bleaches” when exposed to light. 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. 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. 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!).
To develop your blot, you simply soak the surface of the blot in developing solution for 1-2 minutes. Then, seal it up in plastic wrap to keep it from dripping, and trot over to the developing room. The developing room should have a red light, which is okay to keep on while handling film. 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. Every protein-antibody combination has a different optimal exposure time, but you’ll usually want to expose the film for 1-10 minutes. Open the cassette carefully to avoid sliding the blot and film relative to each other (which will result in blurry bands). 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. I’ve seen these around, but I’ve never actually seen anyone use them!
Developing 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.
Using a CCD imager is the more contemporary, hands-off way to image a western blot. These imagers contain a detector that translates the chemical signal (resulting from the HRP-ECL reaction described above) to a digital image. 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. To prepare your blot for a CCD imager, you follow basically the same procedure as you would use when prepping for x-ray film. 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. 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.
CCD 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. 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. These imagers are quite pricey, however – many departments will purchase one for all of their investigators to use. In addition, some imagers require specialized developing reagents, as discussed below.
Things to consider
Whether you use x-ray film or a CCD imager to image your blots, there are a few parameters to take into consideration. Probably the most relevant concern is the sensitivity of the technique you’re using: how bright is your signal, and how difficult is it to detect? The strength of the signal will depend on a lot of variables, including how much protein you have and what concentrations of primary and secondary antibodies you’ve used. If you’re using film, then the surface of the blot is pressed right up against the film, so any signal that’s coming out will be detected. If you use an imager, however, the light has to travel from the imaging stage to the detector, which is often a distance of 12 inches or more. This means that a signal of comparable strength will have to be exposed for a longer time to yield the same end result (or image). For this reason, companies that manufacture CCD imagers often recommend special developing solutions that will generate a brighter signal.
For a detailed comparison of these two different imaging modalities, take a look at this previous article.
For more info on Western blotting, check out this video from Agrisera.