How to detect your target proteins using chemiluminescent detection reagents

Chemiluminescent detection has been the method of choice for western blot analysis because of its high sensitivity, high signal-to-noise ratios, and versatility for use with film or digital imagers. Using this approach, a membrane is probed with an antibody that is conjugated to an enzyme, usually HRP, and a chemiluminescent substrate. Majority of chemiluminescent HRP substrates are two-component systems consisting of a stable peroxide solution and an enhanced luminol or acridine solution. When incubated with a blot to which HRP-conjugated antibodies or other probes are bound, a chemical reaction emits light at 425 nm. This light can be captured with x-ray film, CCD camera imaging devices, or phosphorimagers that detect chemiluminescence. Thermo Scientific SuperSignal Chemiluminescent HRP Substrates provide a range of convenient and reliable solutions for western blot analysis that vary in terms of detection sensitivity and signal duration. And while commercial ECL substrate kits vary in sensitivity, they all follow a same general protocol. It is important to scale your antibody dilutions based on the sensitivity of the substrate being used. Using higher dilutions of secondary antibodies with higher sensitivity substrates will provide better signal-to-noise ratios. To prepare the working solution, first combine the luminol or acridine solution and the peroxide solution at the ratio indicated by the manufacturer. Make a sufficient volume of working solution to ensure that the membrane is completely covered and never becomes dry. Use 0.1 mL of working solution per square centimeter of membrane. For a single mini blot, that’s about 8 mL. Next, pour the solution onto a membrane that has been probed with an HRP-conjugated antibody. For optimal results, use a shaking or rocking platform during incubation steps. After incubating the membrane with the substrate for three to five minutes, remove excess substrate. Wrap the blot in plastic wrap to prevent it from drying. The blot can now be exposed to x-ray film or imaged on a digital CCD-based imaging system. Digital imagers are rapidly replacing the use of film for western blot detection because they are more sensitive, easy to use, and have a wider dynamic range of detection than film. To learn how to capture and analyze images using the Invitrogen iBright imaging systems, watch the how-to video at thermofisher.com/iBright. Learn more information about SuperSignal chemiluminescent substrates at thermofisher.com/chemisubstrates