Western blotting is an analytical technique used to detect the presence of a protein within a complex mixture of proteins such as a tissue extract. Western blots provide a relatively quick means of comparing the levels of a protein of interest between samples, or detecting the presence of posttranslational modifications such as phosphorylation.
In a Western blot, proteins separated by electrophoresis are transferred to a membrane, and the protein of interest is visualized by probing the membrane with an antibody (the primary antibody) that recognizes the target protein. A secondary antibody, conjugated to an enzyme or other label to enable detection, is then allowed to bind to the primary antibody and the blot is imaged.
Presently the most commonly used detection method for Western blotting is chemiluminescence imaged with film, due to high sensitivity and the availability of secondary antibodies conjugated to horseradish peroxidase.
Reproducible Quantitation of Proteins in Chemiluminescent Westerns
Chemiluminescence produces a low-light signal and is traditionally detected using film. Digital imaging is an attractive alternative to film because it has a substantially larger linear range, and because a digital workflow provides increased reproducibility of imaging conditions and the ability to store and share data.
Recent improvements in charge-coupled device (CCD) camera technology, combined with the development of chemiluminescent substrates optimized for digital imaging (ChemiGlow® from Alpha Innotech), have allowed digital images to meet the performance of film for chemiluminescent Western blots. Figure 1 shows duplicate Western blots imaged for 10-second exposures with either the FluorChem® Q CCD imaging system or with film.
The greater linear dynamic range provided by digital imaging allows more quantitative data to be obtained from Western blots. Figure 1 demonstrates the improved linear range that is obtained with digital imaging relative to film. For the digital image, band intensities are linear over the entire protein range, from 0.01 ng to 5 ng (Figure 1B and expanded scale in 1C). In contrast, the film image (Figure 1D and expanded scale in 1E) has a linear dynamic range approximately two orders of magnitude smaller.
Digital imaging also improves laboratory workflow. Imaging protocols can be saved, and imaging times and lighting conditions controlled precisely, allowing improved reproducibility between experiments. Digital images can also be saved and used directly for analysis and publication, whereas film images generally must be scanned for further analysis, a process that can introduce additional artifacts. Digital imaging is also more cost-effective than film, since it does not require a darkroom or film, and is a more green imaging alternative since it avoids the toxic chemicals involved in film developing.