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Imaging Chemiluminescent Western Blots

Use the Eagle Eye II still video system to directly acquire images from chemiluminescent blots

Michelle Cayouette Jane Moores

The Eagle Eye II still video system is well suited for direct imaging of chemiluminescent Western blots. The light-tight cabinet is ideal for the longer exposure times necessary to capture chemiluminescent signals. Also, the systems charge-coupled device (CCD) camera efficiently captures those wavelengths of light emitted by common chemiluminescent substrates. A comparison of chemiluminescent substrates from two manufacturers showed greater than a 10-fold difference in signal intensity. This difference indicates that by carefully choosing the appropriate chemiluminescent substrate for their system, researchers using the Eagle Eye II still video system to image chemiluminescent Western blots may be able to enhance their results.

The Eagle Eye II still video system is a powerful digital documentation and analysis tool for scientific researchers. The system combines quick and easy image acquisition with software processing functions, such as molecular weight determinations, densitometry calculations, and colony or plaque counting.1 Because of its light-tight cabinet and optimized camera sensitivity, the system also efficiently captures images directly from chemiluminescent experiments.2 Although the Eagle Eye II still video system can capture light emitted from the wide variety of substrates typically used in chemiluminescent Southern, Northern and Western blotting techniques, we have found that the substrate choice can have an impact on the quality of the resulting images.

Chemiluminescent Western Blots: Substrate Comparison

Chemiluminescent substrates were compared using the Eagle Eye II still video system to capture an image following Western blot analysis. Dilutions o f Stratagenes purified Epidermal Growth Factor Receptor Kinase Domain (EGFRKD) protein were fractionated through duplicate SDS-PAGE gels, electrophoretically transferred to nitrocellulose membranes and subjected to Western blot analysis using Stratagenes PY20 Monoclonal Antibody Kit. Following primary and secondary antibody incubations, blots were immersed in either ECL immunoassay signal reagent or SuperSignal ULTRA chemiluminescent substrate3 using the manufacturers instructions. Membranes were then covered in plastic wrap and placed side by side within the cabinet of an Eagle Eye II still video system. After the optical filter was removed from the filter holder of the system, a 1-minute exposure was taken with the cameras aperture fully open (f1.2) to ensure maximal signal capture efficiency.

figure 1

The Eagle Eye II still video system successfully captured light emitted from both chemiluminescent substrates (figure 1). After a 1-minute exposure, 9.4 ng of purified EGFRKD protein was detected when using SuperSignal ULTRA substrate, whereas 37.5 ng was detected when using ECL substrate. One way to determine relative sensitivity of the substrates is by comparing the integrated density measurements of bands.1 As long as pixels within analysis areas are not saturated and the dimensions of the analysis areas are kept constant, band concentration is directly proportional to integrated density. Consequently, under similar experimental conditions, bands of the same concentration will have equivalent integrated density measurements and vice versa. Densitometric analysis using EagleSight software version 3.1 revealed that the 18.75-ng band that resulted from using SuperSigna l ULTRA substrate had an integrated density measurement equivalent to a 216-ng band using ECL substrate, based on the equation of a standard curve generated when all four of the visible ECL bands were used as standards. This equates to an 11.5-fold increase in sensitivity observed when using SuperSignal ULTRA substrate compared to ECL reagent. These results suggest that researchers using the Eagle Eye II still video system to directly image filters from chemiluminescent Western blots may be able to increase sensitivity or decrease exposure time by using SuperSignal ULTRA substrate.


The Eagle Eye II still video system features a light-tight cabinet and a CCD camera that is sensitive to wavelengths of light emitted by common chemiluminescent substrates. These features make this system well suited for directly acquiring images from chemiluminescent Western blots. Western blot analyses performed using two different chemiluminescent substrates indicated that some substrates may perform better than others when used with the Eagle Eye II still video system. Consequently, researchers using the Eagle Eye II system to acquire images from chemiluminescent Westerns blots may be able to improve results by carefully choosing the chemiluminescent substrate.


The authors wish to thank Kerstien Padgett for helpful discussions concerning Western blot analysis.

  1. Cayouette, M., Steege, M., and Moores, J. (1997) Strategies 10: 21-23.

  2. Gackstetter, T., et al. (1994) Strategies 7: 57-59.

  3. Mattson, D.L. (1996) Previews 1: 2-3.



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