WesternBreeze™ Chemiluminescent Kits, from Invitrogen, detect proteins that have been immobilized on membranes (Nitrocellulose or PVDF) following Western transfer, or directly from solution (dot blots). Detection is accomplished with a ready to use CDP-Star chemiluminescent substrate for alkaline phosphatase. Protein bands can be captured either by X-ray film or a CDP-Star compatible imaging system.
We used the Anti-Rabbit WesternBreeze™ Chemiluminescent Kits for detection of cell wall proteins from yeast. Samples were run on a 8 cm x 8 cm mini gel (NuPAGE™ 4-12% Bis-tris gel; 1 mm x 12 well). Proteins were detected using a Bio-Rad imaging system as well as X-ray film.
We chose WesternBreeze™ Chemiluminescent Kits because these great kits provide all the solutions you need for detection of a Western blot, all in one kit. Solutions are pre-mixed and optimized to provide reproducible blots, time after time, using just one fast, and simple protocol. WesternBreeze™ Chemiluminescent Kits also offer high specificity, clean background, high sensitivity (femtogram levels detectable) long-lasting signal (up to 5 days), results in less than 3 hours and generation of a permanent photographic image.
WesternBreeze™ Chemiluminescent Kits include blocking solutions, primary antibody diluent, ready-to-use secondary antibody solution (anti-mouse, anti-rabbit or anti-goat depending upon which kit you purchase), ready-to-use CDP-Star chemiluminescent substrate, wash solutions, incubation trays, pre-cut filter paper, polyester sheets for even substrate development on the membrane. Each kit contains enough reagents for 20 blots. The kits should be store at 4ºC, stable for 6 months when properly stored.
The WesternBreeze™ Kit produced a strong signal with low background the first time I used it, with no need for optimization of the secondary antibody concentration.
Protein detection on a membrane is more sensitive than in-gel detection. Since the membrane detection is more sensitive, less HRP-conjugated binding protein (streptavidin or antibody) is required to produce a strong chemiluminescent signal. For this reason, I feel that WesternBreeze™ Kit is best option for protein detection.
There are 6 different WesternBreeze™ Kits. The WesternBreeze™ blocking and wash solutions are also available for individual sale. These solutions play an important role in effectively reducing non-specific binding and background in a Western blot. These preformulated, easy to dilute solutions, provide the researcher with the flexibility to optimize the detection procedure while enjoying the benefits of the WesternBreeze™ system.
Ali A. Latiff, PhD
Researcher
Case Western Reserve University
Biomedical Research Department