Recently, I had the joy of using the Amersham ECL Advanced™ Kit for the screening of Clara Cell proteins (CC10) on Western blots from mouse Clara Cell isolates following exposure to styrene oxide racemic (i.e. a mixture of styrene oxide R and styrene oxide S). The ECL Advanced™ in conjunction with a primary polyclonal from Santa Cruz Biotechnology (rabbit
(FL-96): SC-25555) proved to be an interesting and rather challenging experimental exercise. The polyclonal antibody generated high background due to cross-reactivity with other proteins. However, with the proper conditions, a specific reaction and signal was found. The relatively high likelihood of background when using ECL Advanced™ also contributed to the effort required to generate a clear, specific signal. This combination of a very reactive polyclonal primary and ECL Advanced™ is not for individuals who are looking for an easy, robust assay or a simple cookbook detection of Western blots.
I spent a fair number of experiments testing various wash volumes and times, dilutions and incubation times of primary and secondary antibodies in order to determine the conditions which would show specific reactivity of the polyclonal with CC10. I also screened the primary in an ELISA; this was how I found that the polyclonal primary had a rather high cross-reactivity with a large number of proteins. This information helped to determine the proper conditions for viewing and exposing the film to detect and highlight the protein of interest. Of course, ECL Advanced™ is more easily used with specific primaries. However, once the proper conditions are set, it is easy to use and works rather well.
The ECL Advanced™ system had a significant amount of pep and zip to it with respect to signal generation. I had significant levels of background signal so initially, I used varied incubation times with the ECL Advanced™ reagent as well as varied film exposure times. I recommend the secondary which I used because of its low background and high specificity. I used an HRP conjugate from Zymed (now owned by Invitrogen): Subclass balanced goat HRP Cat. 62-6120.
Once I had determined the optimal conditions for detection of my Westerns, I scanned the film then and fixed regions were selected with UN-SCAN-It Densitometry Software. The regions were then measured and the number of pixels was counted with respect to area and gray levels. These were then converted in number of pixels per 50000 Clara Cell Isolates. Relative changes were noted with respect to controls.
In the figure, note the cross-reactivity to molecular weight markers in lanes 2 and 10. Lanes 4, 6 and 8 are from three different mouse Clara Cell Isolations. Lanes 11 and 13 are Biovender Human CC10 homogenate. The blot is before the stringency was changed to recognize only mouse CC10 protein sequences.
In summary, I would be delighted to have the opportunity to use ECL Advanced™ once again for either Western blot, dot/slot blot, Southern and/or Northern blot application. ECL Advanced™ is indeed a powerful and useful tool for the evaluation and analysis of your research work.
Former Scientist II
Division Biochemical/Diagnostics
Boehringer Mannheim