ECL Plus™ Western Blotting Reagents from GE Healthcare (formerly Amersham Biosciences) provide reliable and sensitive detection of proteins by immunoblotting. This system utilizes chemiluminescence technology for the detection of proteins. It consists of a lumigen PS-3 acridan substrate, which is converted to an acridinium ester intermediate when catalyzed by HRP. The ester intermediate reacts with peroxide in alkaline conditions and emits light, which can be detected by autoradiography (film) or CCD camera. The kit includes Solution A (substrate solution containing tris buffer) and solution B (acridan substrate solution in dioxane and ethanol); the 2 solutions are then mixed in 40:1 ratio.
While HRP-based chemiluminescence kits are available from other companies, one reason we chose the ECL Plus™ detection system is that we use PVDF membrane from GE Healthcare. This membrane has been optimized for compatibility with ECL and ECL Plus™, and their performance is good and consistent. We have been using this kit for more than 3 years and have never thought of switching reagents because we have never had any problem. Reagents from Sigma (luminol and indophenol) as well as H2O2 can be procured locally for detection. These reagents are far cheaper than the kit (if we compare the per reaction price), however, one has to compromise on quick results, duration of signal, and sensitivity; all of which are extremely important at times. Another disadvantage of these cheaper reagents is they require a significant amount of secondary antibody. For example, we use ECL Plus™ with a 1/8000 to 1/12500 dilution of anti-mouse HRP antibody from GE Healthcare. In contrast, some researchers use a 1/1000 dilution for detection with luminol + indophenol. Hence, secondary antibody is consumed in large amounts.
ECL Plus™ is 4-20 times more sensitive than the standard ECL detection reagents. This quality has its advantages and disadvantages too. For example, when we need to detect low abundance proteins, we use ECL Plus™; however when protein is abundant, then the standard ECL reagents perform well. When detecting abundant protein with ECL Plus™, we have found that the signal is so strong that comparing signals from experimental and control samples is difficult. A definitive advantage of the ECL Plus™ reagents over standard ECL reagents is the extended signal duration. For example, developing a blot with ECL over time intervals like 1, 5, 10, 20, and 60 min (total time 96 minutes) gives an increasing signal intensity, but then the signal decreases. With ECL Plus™, the signal remains strong after 2 hours.
We use ECL Plus™ for the detection of Intermediate Filament (IF) and associated proteins in cell lines, human and rat tissues. We have also used it for certain cell cycle associated proteins. One aspect of our research involves modification studies on IF as a result of cell transformation. After blotting the membrane once, we strip the blot for re-probing with modification-specific antibodies to confirm the presence/absence/extent of modification. We detect our blots by exposure to X-ray film. ECL Plus™ combined with PVDF from GE Healthcare is rigid enough to resist stripping at least 5 times; there is a significant decrease in signal after the 5th stripping. However, this is our personal experience with two antibodies; results can vary with different antibodies. In any case, we found ECL Plus™ satisfactory for our experiments.
The ECL Plus™ reagents are easy to use. However, there are some general precautions which need to be taken when using the kit: Avoid exposure of ECL Plus™ reagents to light and make sure to use enough reagents to completely cover the membrane. This sometimes poses a little problem in dark, as it is difficult to know whether the whole membrane is covered with reagent; hence, one tends to use little more ECL Plus™ reagent than might otherwise be necessary. Also, the kit is not stable over long periods of time; the signal appears to be weakened in kits that are more than one year old. However, we have used kits more than 1½ years old and they have worked. The only improvement that could be made in this product is reduction in cost.
Deepak Kanojia
Senior Research Fellow
Cancer Research Institute, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), TMC
Biochemistry and Cell Biology