Bart's Cookbook-- Using ECL instead of iodinated protein A ECL is appealing because it is fast and does not employ radioactivity. It is however inferior to the use of iodinated protein A in a number of ways. First, it is notoriously non-quantitative. In contrast, analysis of iodine-blots with a phosphoimager yields somewhat quantitative data that can be improved by analyzing several dilutions of samples of interest. Second, the technique is prone to artifactual signals arising from abundant proteins on the blot. This manifests itself as a higher non-specific background when weak signals are being examined. This is most apparent when analyzing tyrosine phosphorylation in cell lysates. Tyrosine phosphorylation occurs at a low level in untreated normal cells. When analyzed by ECL, faint signals can be seen to arise from the abundant proteins in the lysate. This obscures the weak specific signal arising from such stimuli as T cell or B cell activation. Although the background may be slightly lower with nitrocellulose than Immobilon-P, most people prefer Immobilon. Azide inhibits HRP. Don't use it in the solutions containing the HRP-conjugated detection reagent. Procedure. 1. If necessary, rewet the blot in methanol and then wash it in rinse buffer. 2. Block for 30 min in 3% BSA in rinse buffer. 3. Incubate 1 hr in primary antibody in 3% BSA in rinse buffer. 4. Wash 2x 10 minutes rinse buffer, 1x 10 minutes 0.2% Tween-20 in rinse buffer, 2x 5 minutes rinse buffer. 4a. Alternatively, wash four times for 10 minutes with 200 micromolar Tris-HCl, pH 7.5, 500 millimolar NaCl, 0.05% Tween-20. This procedure is currently the most popular. 5. Incubate 1 hr in an appropriate dilution of HRP-conjugated detection reagent in blocking buffer. Currently, the anti-mouse antibody is used at 1:5000, the anti-rabbit antibody at 1:10,000, and the HRP-protein A at 1:20,000. 6. Wash as in step 4 or 4a. 7. Then, in the smallest clean tupperware dish into which your blot will fit, mix equal volumes of ECL sol'n A and sol'n B (they recommend 0.125 ml sol'n per cm2 blot, but you can use less if your blot is large). 8. Using forceps, remove blot from rinse buffer, let drain briefly from an edge onto a paper towel, place protein side up in ECL sol'n. Agitate (to make sure blot is continuously covered by the sol'n) for 60 sec. 9. Using forceps, remove blot, drain as before, and place protein side up on a sheet of transparent acetate that is used for the overhead projector. Place another sheet of acetate on top. 10. Initially try a 15 sec exposure and adjust time after developing. (Try pressing a piece of film down on the blot with a glass plate to get even pressure.) Back to Table of Contents