SuperSignal West Pico Chemiluminescent Substrate Kit From Pierce

Western blots are commonly used in molecular biology to detect the presence and amount of protein in a particular tissue, lysate, cell, etc. The sample is loaded onto a polyacrylamide gel and run using electrophoresis. After transfer of the separated proteins to a membrane, the blot is blocked, washed and incubated with a primary antibody specific to the protein of interest. A secondary antibody, usually either conjugated with an AP (alkaline phosphate) or HRP (horseradish peroxidase) is then used to allow visualization of the protein bands. Chemiluminescent detection of HRP-labeled antibodies is much more convenient than AP color development, as the developing time can be varied and tested and the blots can be stripped and reused.

The Super Signal West Pico Kit from Pierce contains a highly sensitive chemiluminescent substrate that allows detection of the HRP-conjugated secondary antibody. It consists of two solutions – a luminol/enhancer solution and a stable peroxide solution. These can be used in either the light or the dark, eliminating the worry of diminishing signal in room light. The solutions are mixed in a 1:1 ratio before use and this mixture is stable for up to 8 hours at room temperature. This mixture is then applied to the blot, and incubated for 5 minutes. Exposure using X-ray film or photography using an imaging system can then be performed.

I have used this kit many times and have not had any problems. It is able to detect even small amounts of antibody or signal. Pierce claims pictogram-level sensitivity, as the name implies. It works well with the secondary HRP antibodies that I have used, although adjustment of the 2° antibody dilution (usually 1:20000 to 1:50000) may be necessary to increase or decrease the background signal. The development time is very fast and the signal lasts for a long time, up to several hours. This allows plenty of time to optimize the exposure and desired signal strength for the blot. One of the most convenient things is the ease of stripping the blot after development. Pierce also sells a Western Blot Stripping Buffer that works very well in as little as thirty minutes. It strips the antibody entirely, without removing the protein from the blot. This allows one to use a blot 3+ times with several antibodies; very important when tissue samples are scarce. While there may be a few kits that are more sensitive out there, I have been very happy with this SuperSignal West Pico Chemiluminescent Substrate Kit and would highly recommend it for developing Westerns.