Western blotting is an essential method for the detection and quantification of proteins from cell and tissue lysates. There are several different Western detection methods available depending on the sensitivity needed: colorimetric, chemiluminescent or labeling the proteins with radioactivity. The most sensitive, inexpensive and fast method is chemiluminescence. The light-emitting enzymatic reaction used in chemiluminescent detection is based on the oxidation of a luminol-based substrate by horseradish peroxidase. The horseradish peroxidase is coupled to the primary antibody, which detects the antigen, or to the secondary antibody, which binds to the primary antibody. Light emission can easily be captured and visualized by exposing the blot to film or the CCD camera of an imaging system.
To detect very low abundant proteins/antigens on a Western blot, Pierce has developed the SuperSignal® West Femto Maximum Sensitivity Substrate, which is sensitive enough to detect proteins in femtomolar concentrations. In addition, it limits the amount of antibody necessary for detection. The detection procedure is easy and fast. The substrate is prepared by mixing equal volumes of the stable peroxidase solution with the luminal/enhancer solution and incubating the blot for 5 min in the solution before exposure to film or imaging system. The substrate is provided as a trial kit with 20 ml of each detection solution, or as kit containing either 100 ml or 200 ml of the detection solutions and 1 ml of an HRP-coupled goat anti-mouse antibody and an HRP-coupled goat anti-rabbit antibody.
Since I was using the trial kit for the first time, I titrated the concentrations of the primary antibody, the HRP-conjugated secondary antibodies available in my laboratory, and the amount of protein loaded onto the gel/blot. It is extremely important to do this before using the kit since concentrations suitable for most chemiluminescent kits will result in overexposed bands with high background; this is due to the ultra-sensitivity of the SuperSignal West Femto Substrate. In addition, it is important to use high quality reagents for preparing the solutions and clean containers to incubate the Western blots. Titrating the antibodies is done by spotting 2-3 different protein concentrations (for each antibody dilution) on the membrane, drying the membrane and re-hydrating (PVDF in methanol and buffer, nitrocellulose in buffer) before blocking and exposing it to the different antibody concentrations as with a standard Western blot. It is good to try 2 different very dilute concentrations of the primary (1:3000 and 1:5000 (mouse) or 1:5000 and 1:20000 (rabbit)) and second antibodies (1:200000 and 1:500000). For an antigen of 55 kD, I used 1, 2, 5 µl of total a protein solution containing 0.5 µg/ml protein, a primary rabbit antibody concentration of 1: 3000 and 1:10000 (antibody concentration 0.5µg/ml) and a secondary antibody concentration of 1:100000 and 1:300000 (Pierce H+L specific IgG antibody, concentration 1 mg/ml). Lower concentrations of the secondary antibodies (1°:1:3000 and 2°:1:500000) resulted in good visible bands with less background while higher antibody concentrations resulted in dark bands and high background. When I blocked with a 5% low-fat powdered milk/PBS/0.05%Tween-20 solution, I obtained speckled blots, but higher Tween-20 solution (0.1%) and longer washes improved the background. Between the antibody incubations, I extensively washed the blots while changing the solution 6 times in 30 min; after the second antibody incubation, 10 times in 60 min, which also helped reducing the background.
The SuperSignal® West Femto Maximum Sensitivity Substrate is an easy to use detection system for detection of very low abundant proteins in Western blot. The manual provided in the kit contains information about important adjustments in the protocol, like titrating the protein and antibody concentrations or using extended washing steps and ultra-pure reagents. Although this kit requires some additional work to optimize the detection, it is very useful for detection of very low protein amounts in cell and tissue lysates.