Western Blot Imaging and Analysis

There are several methods for capturing data generated from chemiluminescent Western blots, including x-ray film cooled CCD cameras and phosphorimagers that detect chemiluminescence. Cooled CCD cameras, which offer the advantages of instant image manipulation, greater resolution and larger dynamic range than film, also eliminate the need for a darkroom and film-processing equipment. While X-ray film can be used to obtain semi quantitative data, digital imaging is more sensitive because of the broad dynamic range of detection, allowing researchers to obtain quantitative data from Western blots.

Although electronic data capture with digital cameras and imagers is growing in popularity most of the data obtained from Western blotting with chemiluminescence is still captured on film. Often, it is necessary to expose several films for different time periods to obtain the proper balance between signal and background. The goal is to time the exposure of the membranes to the film so that the desired signal is clearly visible while the background remains low.

This is difficult to accomplish because the process cannot be observed and stopped when the desired endpoint is reached. If the film is not exposed long enough (underexposed), the signal will not be visible. If the film is exposed too long (overexposed), the signal may be lost in the background or separate bands may become blurred together. An overexposed film can be “fixed” by incubating it in Thermo Scientific Pierce Background Eliminator Solution (Product # 21065), which effectively decreases the background without altering the integrity of the data. This is done at the lab bench while watching the film and the process can be halted when the signal is clearly visible and background is at a minimum.

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