Invitrogen’s WesternBreeze® Chemiluminescent Western Blot Immunodetection System is a non-radioactive detection method for antigens that have been immobilized on nitrocellulose or PVDF membranes by Western transfer. The system uses alkaline-phosphatase conjugated secondary antibodies in combination with a chemiluminescent substrate for alkaline phosphatase to produce a signal that can be captured on x-ray film or by other imaging systems.
Traditional Western detection employs tris buffers for diluting primary and secondary antibodies and for washing steps. The blot is incubated in reconstituted dry milk or BSA to block nonspecific binding, then washed and incubated for ~ 1 hour in an appropriate dilution of primary antibody. The blot is washed extensively, incubated for ~1 hour in a dilution of species-specific secondary antibody (usually anti-mouse or anti-rabbit) and washed again prior to detection. Secondary antibodies are often conjugated with horse-radish peroxidase (HRP) or alkaline phosphatase (AP) to allow chemiluminescent detection with substrates for HRP or AP. Antibody dilutions are made in buffer, and can usually be reused up to six times, an advantage if the primary antibody is scarce or expensive. Optimization of antibody concentrations may be necessary to obtain sufficient signal or reduce background.
The WesternBreeze® system differs from more traditional systems in that it provides a proprietary blocker/diluent solution for blocking nonspecific binding and making primary antibody dilutions, and the secondary antibody comes diluted and ready-to-use. Invitrogen claims several advantages for this system, including high sensitivity and low background with no need for optimization of the secondary antibody; reduced time to perform the protocol compared to traditional Western detection; and a chemiluminescent signal lasting up to 24 hours compared to just a few hours with other systems.
In my hands, the WesternBreeze® system produced a strong signal with low background the first time I used it, with no need for optimization of the secondary antibody concentration. However some important disadvantages were not apparent from the product literature, most notably that primary antibody dilutions cannot be reused: an attempt to reuse the primary antibody dilution, and to use previously made dilutions of primary antibody in non-proprietary buffer, resulted in unacceptably high background. With regard to time, the product literature claims a savings of up to 17 hours over conventional Western blotting. Most of this savings is illusory because 15 hours comes from the blocking step, which in the conventional protocol can be performed either for 1 hour at room temperature or at 4ºC overnight. In reality, blocking overnight can be a convenience if you spent the day preparing your samples, running a gel, and performing Western transfer. A modest savings in time is achieved by using ready-made reagents such as the wash solution included in the kit. The claim of a longer lasting signal also proved unjustified in my hands; several attempts to expose films overnight resulted in no detectable signal in the morning. Additionally, with the WesternBreeze® system you must purchase a separate kit for every species of secondary antibody you want to use, instead of simply buying the conjugated antibody.
If the manufacturer’s directions are followed closely, the WesternBreeze® system is sensitive and reliable. However, because primary antibody dilutions cannot be reused, and a separate kit is needed for each species of secondary antibody, I find other Western detection systems more economical and convenient to use.
Kimi Nishikawa
Graduate Student
State University of New York at Albany