SuperSignal West Femto Chemiluminescent Substrate From Thermo Scientific Pierce

Chemiluminescence is one of the most popular and widely used methods for detecting proteins on Western membranes. Chemiluminescence uses horseradish peroxidase (HRP) enzyme-labeling to detect proteins of interest. The HRP enzyme reacts with peroxide to emit light upon its decay; thereby, allowing visualization of the desired protein.

The SuperSignal West Femto Chemiluminescent Kit from Pierce is available in two sizes (100 and 200 mLs) plus a trial size (20 mLs), so the product can be tested and optimized while being cost-effective. The substrates are good for one year when stored at 4ºC and six months when stored at room temperature. The SuperSignal West Femto Kit is the most sensitive detection substrate that Pierce currently offers. Pierce maintains it can detect protein amounts in the mid-zeptomole (10^-20) range.

To use the substrate, first mix the enhancer and the peroxide solutions at a 1:1 ratio. Care must be taken to ensure the stock solutions do not come in contact with each other (cross-contamination can cause inactivation of the reagents). Following the appropriate antibody incubations and washes, the membrane is incubated with the detection solution for five minutes with gentle agitation. The peroxide solution is sensitive to light but a small amount of laboratory lighting during the membrane incubation is acceptable. It can then be exposed to film or imaged with a CCD imaging system.

I used the Pierce SuperSgnal West Femto Chemiluminescent Substrate Kit to visualize proteins from cell lysates. After SDS-PAGE, I transferred the proteins to a PVDF membrane and followed standard Western Blotting protocol. I blocked the membrane with 5% non-fat milk in TBS/Tween (0.01%) for one hour at room temperature. Then I incubated the membrane with primary antibody overnight at 4ºC. I washed three times for 10 minutes with TBS/Tween (0.01%), after which I incubated with secondary antibody (1:25,000) labeled with HRP. Then I washed three times again for ten minutes with TBS/Tween (0.01%). I applied the substrate for five minutes, according to the Pierce protocol, and then removed the excess moisture before enclosing the membrane in plastic wrap and imaging.

Short exposures should be taken first to judge the effectiveness of the substrate and antibody in combination. Follow with longer exposures until the optimal exposure time is obtained. The Pierce protocol claims that the SuperSignal West Femto Substrate has a signal duration of eight hours, but a twenty minute image was sufficient for my membrane.

I was very pleased with the high sensitivity of the substrate and its overall perfomance. The protocol was easy to follow and I only needed a small amount of solution and minimal antibody; thereby, it proved to be cost efficient. I saw no obvious differences in the performance of the substrate when using a nitrocellulose versus a PVDF membrane. However, if the antibody concentration is not optimized, there is a tendency for extra background. Pierce offers a detailed troubleshooting guide and an antibody dilution guide to help facilitate the optimization of the SuperSignal West Femto Substrate. Overall, I was satisfied with the SuperSignal West Femto Chemiluminescent Substrate and will continue to use it for the high sensitivity detection of proteins.