The use of non-radioactive chemiluminescent substrates to visualize proteins on a western blot makes the entire process both sensitive and simple. The substrate that I have been using is the Western Lightening Chemiluminescence Reagent Plus Kit from Revvity. This substrate takes advantage of a chemical reaction in which the enzyme horseradish peroxidase oxidizes the substrate luminol, which, in the presence of phenol and hydrogen peroxide, causes an intense light emission with a maximum wavelength of 428nm. The use of an enhancer in this product increases the emission approximately 1000-fold. According to the manufacturer, the Western Lightening substrate can be used to detect as little as 1-10ng of protein.
I have used the Western Lightning kit numerous times for western blots (using peroxidase conjugated secondary antibodies) on nitrocellulose membranes and have consistently gotten clean; clear pictures with minimal, if any, background. The protocol is extremely simple; mix the reagents (the substrate and the enhancer) 1:1 and allow the reagents to stand on the membrane for 1 minute. After the final wash (to remove excess secondary antibody) I take the membrane, blot the corner with a Kimwipe, lay the membrane, face up, on saran wrap and simply add enough substrate to cover it. I do not find it necessary to shake the membrane during this incubation. After the 1-minute incubation, I remove the excess reagent by wicking it off with a Kimwipe on the corner of the membrane, seal it up in saran wrap and expose it to film. The reaction can be seen in as little as 10 seconds and can be left on film for up to 2 hours, depending on the concentration of protein. Membranes can also be stripped and re-probed if desired.
Overall, I found the Western Lightning Chemiluminescence Reagent Plus Kit to be an asset to my protein detection. The kit is available in a variety of sizes and has a shelf life of 1 year, although it must be stored at 4ºC. A benefit to this kit lies in the fact that the procedure is simple and results are seen immediately. The signal is strongest in the first few minutes of the reaction, but as it slowly declines, is still detectable for up to about 2 hours.
Karrie Comatas
Research Technician II
Duke University