Enhanced chemiluminescence (ECL) is the most commonly used method for routine protein detection in Western blots. ECL is based on the emission of light during the horse radish peroxides (HRP)- and hydrogen peroxide-catalyzed oxidation of luminol. The emitted light is captured on film or by a CCD camera, for qualitative or semi-quantitative analysis.
The ECL™ Western Blot Detection Reagents are sold as a two component kit containing a bottle of luminol substrate and a bottle of peroxide solution. The solutions should be equilibrated to at room temperature and then mixed in equal volumes directly before use. For longer storage, the pre-mixed solution should be kept at 2-8°C, but equilibrated to room temperature before use. Different sizes are available; kits are available that allow detection of 1000, 20000, 4000, 6000 cm2 of membrane. The brochure enclosed in the kit provides a brief troubleshooting guide, a description of the principles of ECL detection and a detailed protocol of the optimized Western blot detection procedure.
After using a detection system where the protein signal is visualized by precipitation of the dye DAB for several years, I favored GE Healthcare’s ECL detection reagents because of the higher sensitivity and the ability to re-use the blot for detection of different antigens. I used this kit for years in different laboratories in several applications (e.g. detection of cytokines and adhesion molecules in total protein lysates from mouse spinal cord and detection of myelin proteins in homogenates of mouse sciatic nerves). The ECL™ Detection System covers the detection of a wide range of protein concentrations and can therefore, be used to detect low abundant as well as high abundant samples on the same blot. This can be achieved by either cutting the blot to probe with different antibodies at the same time or by stripping and re-probing the blot with a different antibody. This method allows the comparison of an unknown protein to a house-keeping gene in the sample for semi-quantitative analysis and comparison of the antigen concentrations in different samples, e.g. stimulated versus non-stimulated protein samples. The kit is also very useful when small and precious protein samples are used (like in my case, an injured versus an uninjured sciatic nerve from the same mouse) and several protein markers have to be determined in the same sample on the same blot.
I followed the manufacturer’s protocol. I used a PVDF membrane because it is more resistant allowing blots to be stripped several times. Since I used a variety of primary and secondary antibodies from different vendors, I needed to adjust their concentrations carefully because of the high sensitivity of the ECL™ detection system. For the incubation of the blot with detection reagent, I placed the blot with the protein side up on a flat plastic lid covered with parafilm before adding the detection reagent to evenly cover the blot. After incubation and removal of excess detection liquid, I placed the blot upside down on a sheet of saran wrap, starting at one corner and lowering the blot slowly to remove any air bubbles. I used first exposure times of 30 sec with a Kodak ML film and then adjusted the time as appropriate. For most signals, exposure times did not vary extensively during the first 30 – 60 min after developing the blot (I never tried longer times), so multiple or prolonged as well as repeated later exposures of the same blot are possible without loosing too much of the signal.
GE Healthcare offers a variety of other ECL Detection Reagents with higher sensitivity, but the ECL™ Western Blotting Detection Reagent remains an excellent system for the detection of proteins across a wide range of concentrations with a high sensitivity and without requiring extensive adjustments to the working conditions for your antibodies.