Successful Western detection is contingent on several factors including primary and labeled secondary antibody concentration and specificity, the amount of target protein and the sensitivity of the detection reagent. Consequently, many researchers opt for the use of chemiluminescent detection methods. One potential drawback to the use of chemiluminescent protocols is the number of intervening steps required before a film image of the immunoblot can be produced and analyzed. Where speed, accuracy and generally low background are considered more critical than a publication-quality image, the use of the Promega Western Blue Stabilized Substrate –Alkaline Phosphatase reagent is the reagent of choice.
One of the main goals in the lab is the global proteomic analysis of mosquito midgut glycoproteins that may serve as adhesion receptors for Plasmodium parasites. As part of this effort we tested the specificity of sera from several animals that were immunized with different mosquito glycoproteins by immunoblot. In addition, in an effort to isolate select glycoproteins from complex mixtures by HPLC, we tested several 96-well fractions for the presence of target proteins by immuno-dot-blot. In both cases, Western Blue detection was used largely due to the speed and exceptional sensitivity that is provided by this chromogenic substrate.
Use of the reagent is simple and straightforward. The final wash steps after incubation with secondary antibody are immediately followed by two exchanges with Milli-Q water. The blot is then incubated for a minimum of 5 minutes in a small volume of the stabilized substrate (2-2.5 mls per 7 x 8.5 cm blot). The appearance of very faint bands, corresponding to protein concentrations well below 1 μg/well, is possible. However, in our experience, this has required prolonged incubation (~ 5-7 minutes) resulting in increased background staining. To avoid this, as a rule we load between 3-5 μg of sample per well to increase the signal-to-noise ratio. The same problem was observed when using poor-quality antisera as probe (e.g., low concentration of IgG or poor specificity). We frequently had to increase the incubation period to 10 minutes or more to allow for adequate protein band development. In such cases, band development should be monitored very closely and the reaction immediately stopped with water. The blot can then be air dried on top of filter paper and stored “as is”. The intensity of the bands will fade after 6-7 months so immediate scanning of the blot is strongly recommended. No discernable differences are observed between nitrocellulose or PVDF membranes.
These types of experiments highlight the convenience of the Western Blue Stabilized Substrate from Promega. A relatively small, but added convenience is that storage of the substrate at room temperature conserves valuable refrigerator space in the lab.
Rhoel Dinglasan, Ph.D., M.P.H.
Johns Hopkins Bloomberg School of Public Health
Promega's Western Blue Stabilized Substrate-AP
Quick, inexpensive and easy to use.
Detection sensitivity limits with increased background.
The Bottom Line
Highly recommended as an added reagent for high-throughput immunoscreens.