June 15, 2009 (Vol. 29, No. 12)

CCD Technology Provides High-Quality Data, Reproducibility, and Increased Throughput

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.

Figure 1. Digital imaging surpasses film for linear dynamic range of chemiluminescent data.

Increased Information from Multiplex Fluorescent Westerns

Analysis of multiple proteins using chemiluminescence typically involves stripping and sequentially reprobing blots for each additional target of interest. This method is time-consuming, and uneven stripping of the blot can cause loss of quantitative information. A recent alternative to chemiluminescence is the use of fluorescently labeled secondary antibodies.

Fluorescent detection makes it possible to assay multiple proteins simultaneously with fluorescently labeled secondary antibodies that have nonoverlapping excitation and emission spectra (Figure 2). Figure 3 depicts a two-color Western blot imaged using the FluorChem Q, in which two proteins were detected using SpectraPlex™ secondary antibodies. 

Figure 2. Multiplex fluorescent Western detection

Simultaneous analysis of multiple proteins on a single blot allows a loading control or internal standard to be assayed alongside a protein of interest for each sample, improving the quantitative accuracy of the data obtained from the blot.

Figure 4 depicts a three-color Western blot, in which the detected antibodies were labeled with CyDyes™ (GE Healthcare) and fluorescein (Vector Laboratories). The amount of transferrin (red) in each sample was normalized using a loading control (IgG, blue), and the fold-change of transferrin relative to the first lane was calculated. The calculated results agree closely with the predicted amount of transferrin, as shown in the Table, demonstrating the quantitative quality of the data obtained from fluorescent Western blots.

Modern improvements in digital imaging make it possible to obtain more quantitative and more reproducible data from Western blots. The availability of comprehensive imaging and analysis solutions like the FluorChem Q enables laboratories to acquire data from chemiluminescent and fluorescent blots, as well as from more routine DNA and protein gels.

Figure 3. Multicolor Western blots allow multiple proteins to be assayed on a single blot.

Figure 4. Internal loading controls with multicolor Westerns

Multicolor Western blots provide quantitative data

Mike Collier, Ph.D., is senior applications scientist, Lisa Isailovic ([email protected]) is marketing manager, and Tamara R. Golden, Ph.D., is technical applications writer at Alpha Innotech. Web: www.alphainnotech.com.

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