There is a variety of detection methods available for proteins in Western blots. Chemiluminescence is one of the most sensitive methods; it is based on the enzymatic reaction of horseradish peroxidase (HRP), which is either coupled to secondary or primary antibody. HRP catalyzes the oxidation of a luminol substrate by peroxide, emitting light during the decay to its ground state. The light emission can be either detected by exposing the blot to film or it can be captured with the CCD camera of an imaging system.
The SuperSignal® West Pico Chemiluminescent Substrate from Pierce is a two-component solution: the stable luminol reagent with enhancer and the stable peroxidase reagent. After mixing both solutions in a 1:1 ratio, the blots are incubated for 5 min in this detection solution and then can be immediately exposed to film or CCD camera. Therefore, the detection method is fast and due to the enhancer in the luminal solution, very sensitive. Even though the peroxidase solution is light sensitive, exposure to laboratory light is acceptable during the incubation. The Pierce substrate kit is less expensive than comparable chemiluminescence detection kits from other vendors and is stable: there is no loss of activity when stored at room temperature for one year.
I used the Pierce SuperSignal® West Pico Chemiluminescent Substrate to detect proteins from cell and tissue lysates, gradient fractions or after immunoprecipitations. Since most of my proteins were low abundant in the lysates, I valued the high sensitivity of the system. For higher protein-binding capacity, I used a PVDF membrane to transfer the proteins and blocked the membrane routinely with 5% low-fat powdered milk in TBS/Tween-20 (0.05%) for 1 h at room temperature, followed by overnight incubation with the primary antibody. After 3 washes, I incubated with the HRP-labeled antibody from Pierce (1: 40000 (mouse) or 1:60000 (rabbit)) and washed more extensively (6 x 10 min) again in TBS/Tween-20 (0.5%) to avoid non-specific bands as background. I incubated the blots with shaking and in enough substrate (usually 5 ml for a miniblot) so that the blot was fully covered with solution in order to avoid uneven detection of bands. Before placing the blot between sheets of a clean, dry plastic folder, I drained the excessive detection solution onto a dry Kleenex. I used ultra-sensitive film and only exposed my blots 10-30 sec, or 1-5 min for detection of very high molecular weight proteins. I got very clean and good, visible signals. Using the Kodak 2000MM Multimodal Imaging System, I acquired pictures using exposure times from 1 to 10 min and sometimes also used an accumulated imaging of 3x10 min exposures to capture low signals. During longer exposure times: from 10 to 60 min, I sometimes experienced a decay of the light emission, whereas images were still detectable when I sometimes exposed for 60 min following a 30 min exposure. Pierce claims that the reaction is stable for 6 hours. Since the antibody concentrations were optimized with dot blots prior to their use in an experiment, the difference in detection cannot be traced back to inadequate antibody and/or HRP concentrations. Due to the multiple steps involved, it is difficult to find out what changes might be responsible for the sometimes faster decline in light output: it could be the protein transfer, the primary antibody binding to the protein, the binding of the 2nd antibody, insufficient mixing of the reagent or contamination in any one of the solutions, which might result in degradation of proteins/antibodies. I usually prepared new washing/incubation buffers after experiencing a faster decline in signal.
The SuperSignal® West Pico Chemiluminescent Substrate is a very easy to use, sensitive and fast detection system. I highly recommend the substrate system for most applications since the signals are intense without background when exposed to film for shorter times. For more challenging applications which require longer exposure times, as with low abundance proteins and a less sensitive CCD camera, this system might be less suitable.
The instruction manual in the kit contains an extensive protocol for detection of Western blots as well as a troubleshooting table. Pierce’s website provides a table comparing different chemiluminescent substrates to help in choosing the right one for the application.