Western blotting is a basic technique, used routinely for the detection of specific proteins after separation with denaturing polyacrylamide gel electrophoresis (PAGE). Many developments during the last years have enhanced the efficiency and ease of Western blotting techniques. However, due to the large number of steps that must be accomplished successfully, the whole process is very demanding.
I used Western blotting, along with Enzyme Linked Immunosorbent Assays (ELISAs), in order to detect proteins that were isolated from the serum of mice using various affinity techniques. The proteins were purified in PBS solution. Small samples of this solution were tested for purity with SDS-PAGE and subsequent Western blotting. The proteins were transferred onto a nitrocellulose membrane overnight in a cold room. For labeling and detection of the proteins, I used a primary anti-mouse IgG and a secondary rabbit anti-mouse IgG antibody conjugated with Horse Radish Peroxidase (HRP). Detection was accomplished with the ChemiLucent™ ECL Detection (ChemiLucent™ Peroxide Buffer, Luminol Enhancer Solution and Peroxide Solution) Kit. The emission of light is based on the interaction between luminol and HRP; very low amounts of protein can be detected using this kit.
The kit contains enough reagents for about 5,000 cm2 membrane. The reagents included are Luminol/Enhancer Solution (250 mL), Peroxide Buffer (250 mL) and Peroxide Solution (1.0 mL). After the washing of the membrane, I mixed equal volumes of Luminol/Enhancer and Peroxide Buffer and then I prepared a 1:1000 dilution of the Peroxide solution, according to the manual included in the kit. Usually, I prepared 10 ml of this mixture for a membrane with dimensions 10 cm x 5 cm. This solution was stable at room temperature for a long time. (I haven’t tried to use it after more than 2 hours but for this period of time, it gave excellent signal.)
The membrane was transferred to the ChemiLucent™ working solution in the dark room. For the detection of light emission, I used a Kodak X-ray film. After incubating the membrane with the working solution for about 5 min, I exposed it (protein side facing towards the film) to the film for 30 sec and then according to the signal strength, for longer periods until I had obtained the desired result. The signal remained reasonably strong even after 1 hour of exposure and bands that emitted light with low intensity, were easily identified.
Whenever every step of the Western blot procedure was accomplished successfully, the detection of the proteins using the ChemiLucent™ Kit produced a strong signal with zero background.
The kit, if stored at 4ºC, can remain active for many months. One small drawback is the number of solutions that must be mixed in order to prepare the working solution; other kits contain fewer solutions and consequently, they are easier to handle. The ChemiLucent™ Kit, however, is a very efficient and reliable product and I would recommend it to anyone wishing to get clear results at the end of the long duration of each Western blot procedure.
Graduate Student
Department of Biology
University of Crete