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Imaging Chemiluminescent Western Blots

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
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