Western Blotting, also called protein immunoblotting, is a very commonly used technique to detect proteins in tissue or cell lysates. Briefly, in this technique, the cell homogenate is separated into constituent proteins by PAGE (polyacrylamide gel electrophoresis). The separated proteins are transferred on to a PVDF or nitrocellulose membrane and the membrane is incubated with an antibody specific to the target protein. The membrane is then generally incubated with a secondary antibody that is conjugated to a tag, often an enzyme. This enzyme, when treated with appropriate substrate produces a colorimetric, chemiluminescent or fluorescent reaction which can be captured directly on the membrane, film and/or an imager.
There are several substrate cocktails available in the market for the detection of proteins on the Western blot. It is crucial to choose the appropriate substrate that is sensitive enough to detect the target proteins even in low concentrations and yet not create noise due to non-specific signals. We have used Pierce's Enhanced Chemiluminescent (ECL) Western Blotting Substrate for several years. In our experience, we have found that this kit generates good results with most proteins. However, on occasion, if non-specific binding is not properly blocked or if the primary antibody cross reacts with the blocking agent, heavy background signals were seen. Therefore, it is essential to optimize conditions for each antibody being used. We have detected proteins using this reagent in primary tissues such as human and mouse fetal brain and liver, as well as in several secondary cell lines including neuroblastoma and osteosarcoma lines.
The Pierce ECL Western Blotting Substrate is non-radioactive and highly sensitive. It is a luminal based chemiluminescent substrate and therefore needs to be used and stored in the dark. It is extremely easy to use. It comes with two reagents: detection reagents 1 and 2, which are to be stored at 2-8°C until use. Once the membrane is treated with the blocking agent, primary and secondary antibodies, and washed, reagents 1and 2 are mixed in equal volumes in a separate tube. The membrane is incubated in this mixture for 1 minute in the darkroom and exposed to X-ray film after draining the excess reagent. We have been able to expose membranes treated with this reagent repeatedly to X-ray film in order to get optimal results. Typically with proteins in higher concentrations, we start with a 1 minute exposure and increase or decrease the exposure time depending on the intensity. With certain proteins, such as total kinases, we obtained good images with even 30 second exposures. However, we have exposed up to 30 mins when identifying phosphorylated kinases. Further, we also were able to strip the membrane using Restore Western Blot Stripping Buffer (also available from Thermo Scientific) and use the same reagents to re-probe for other proteins. Since the sensitivity of the reagent is high, we were able to probe even low expressing proteins with small concentrations of the antibody. In general, we have been able to get good results by diluting the antibody a thousand to five thousand fold. This is important since both monoclonal and polyclonal antibodies are generally very expensive. Further, the cost of the reagents is not too prohibitive.
In summary, ECL Western Blotting Substrate from Pierce is an excellent product for detecting most proteins in various tissue and cell culture lysates.
Pierce ECL Western Blotting Substrate from Thermo Scientific
Because the substrate is chemiluminescent, lower concentrations of antibody are sufficient.
Non-specific signals are detected at times due to the high sensitivity of the substrate; therefore proper blocking is crucial.
The Bottom Line
This substrate works well to detect most proteins on a Western blot.