Western blotting is a staple technique used in a variety of labs. Thus efficient and cost effective reagents are very important. And although sometimes newer is better, occasionally it is actually the old standard that works best. The Pierce SuperSignal Substrate system is one of these standards that has stood the test of time. In my lab, this kit has been used for over 10 years for a variety of detection applications including film and an assortment of chemiluminescent imaging systems. We have compared it to other systems and have found the SuperSignal West Pico to be cheaper per square cm of blot and to have greater signal intensity. The increase in signal intensity results in a lower requirement of lysate and antibodies, typical limiting reagents in an experiment. The kit can be stored at room temperature, saving valuable refrigerator space. With only two solutions that need to be combined in equal amounts immediately before detection, the kit is easy to use. The working solution is stable for 24 hours at 4C and I have found it can be used repeatedly, providing maximum use from the kit. In my lab we find that using Supersignal West Pico we get clear reproducible, and most importantly publishable results.
The kit contains two detection reagents: Stable Peroxide Solution and Luminol/Enhancer. These two reagents can be used sparingly, extending the number of blots that can be processed with a single kit. The minimum volume required to cover a miniblot (7 x 8.5 cm) membrane is 1.5 ml (i.e. 0.75 ml of each reagent). By using a smaller volume, you can increase the quantity of miniblots that can be analyzed by approximately three-fold over the manufacturer’s recommendations. For those who are new to Western blotting, Pierce sells the SuperSignal West Pico COMPLETE Kits. These kits include the two detection reagents mentioned above, as well as, secondary antibodies conjugated to HRP, blocking solution and buffer.
As with any two reagent chemiluminescent system, you must prevent contamination between reagents. Once the solutions are mixed, they are only good for 24 hours at 4C, as compared to the individual solutions, which are good at room temperature for 1 year (avoid contamination by using 5 ml pipettes). Additionally, Pierce sells a trial kit so each member of your lab can have their own sample kit with accessories. The 25 ml bottles can then be refilled from the 500 ml stock bottles, thus decreasing the probability of solutions becoming contaminated.
Roseanne Shoshana Greenberg
Graduate Student
Department of Ophthalmology
Mount Sinai School of Medicine
New York, NY