Our laboratory has come to rely on the Pierce ECL Western Blotting Substrate for detection of target proteins on immunoblots for Western blot analysis. This substrate is very sensitive; it is an enhanced, luminal-based, chemiluminescent substrate for the detection of horseradish peroxidase (HRP) on immunoblots. Due to the nature of the substrate, your primary antibody must be bound by an HRP-conjugated secondary antibody. The kit we purchased included Detection Reagents 1 and 2 with each bottle containing 250 ml. The amount is enough, according to the manual, for 4000 cm2
After transferring the proteins from a polyacrylamide gel onto a PVDF membrane, the membrane is blocked. The blot is incubated with primary antibody, washed, incubated with HRP-conjugated secondary antibody and then washed again. The membrane needs to be washed thoroughly after the HRP-conjugated secondary antibody in order to minimize background signal. About 30 min before finishing the wash, Detection Reagents 1 and 2 are mixed at a 1:1 ratio and allowed to equilibrate to room temperature in the dark. The mixture is stable for up to 1 h at room temperature. After the final wash, the blot is covered with the substrate mixture and incubated for about 1 min at room temperature with shaking. The blot is then exposed using X-ray film.
After addition of the ECL substrate mixture, light emission is most intense for the first 3-4 min for most of the antibodies we have utilized, with a decrease in the signal after 5 min. For certain antibodies, such as Anti-HSF-1, I did not get any signal, so I used the Luminol/Enhancer Solution found in the Pierce Chemiluminescent Nucleic Acid Detection Module Kit, which is a lot more sensitive, instead of the ECL Substrate. I applied the Luminol/Enhancer Solution just as I would the ECL Substrate except that I incubated the blot with the substrate for only 1 min due to the increased sensitivity of the Luminol/Enhancer Solution.
We are studying aging using C. elegans as a model system. Currently, I am doing many Westerns using worm extracts, looking at the levels of proteins involved in aging. There are at least three pathways involved in the regulation of aging. One pathway, which we are interested in, involves the insulin/IGF-1 signaling pathway. I am looking at protein levels of certain proteins implicated in this pathway in long-lived C. elegans mutants. I detect actin as a control to ensure that I am loading similar amounts of proteins per well on the polyacrylamide gel. Pierce’s ECL Substrate gives me a very good signal for actin. I can load as little as 2ng of protein extracts and still get an actin signal. There are absolutely no background problems even with minimum washes (i.e. 3 washes with TBS/Tween20 for 5 min after primary and secondary antibody incubations).
Fortunately, using this kit has eliminated problems with high background and the blots can be exposed to X-ray film repeatedly in order to obtain optimal results. The blots can also be stripped of the immunodetection reagents and re-probed. One advantage is that the kit comes with enough reagent for numerous rounds of detection. In addition, the manual that comes with the kit is easy to follow and gives a general Western protocol. On the negative side, the signal seems to decrease pretty quickly for most of my blots, even though the manual claims it should last much longer.