The Chemiluminescent Array Detection Kit is designed to allow the detection of biotinylated probes or targets hybridized to a GEArray cDNA array. The kit contains all the reagents necessary to process at least 12 GEArray nylon membranes. The kit contains the following: Blocking solution (GEAblocking Solution Q), Alkaline Phosphatase (AP)-Streptavidin conjugate, washing buffer (5X Buffer F), alkaline phosphatase assay buffer (Buffer G) and the substrate for the chemiluminescent reaction (CDP-Star Substrate). There are enough reagents for the recommended use although one can get away with using less. The instructions are very clear and easy to follow, even for someone with little lab experience! The result is a very clean membrane with very good signal/background ratio.
We are studying the effect that the anticoagulant and cytoprotective Activated Protein C (APC), in its different forms, has on endothelial cells from different origins. In brief, specific pathway cDNA arrays were used for the analysis of gene expression under different conditions on endothelial cells. After incubation with different stimuli, RNA was extracted from the cells and used to make biotin-labeled probes for the hybridization of the cDNA arrays. For detection of the hybridized probes we used the Chemiluminescent Array Detection Kit as recommended by SuperArray; this allowed us to obtain the raw data for the subsequent analysis by the appropriate software.
We have used this kit over the last 2 years quite regularly and I can say that it performs well every time. Even very new students can very easily follow the instructions and obtain results. The only things to consider are: (1) the right dilution of Buffer F (5X) as it is important not to use it neat due to the effect that could have on the membranes; (2) the amount of wash buffer used (if you use too much you’ll run out of it), normally I use 4 ml per membrane per wash, and (3) to make sure to take Solution Q and Buffer F from the cold in plenty of time to come to room temperature before you begin. I like to take them out of the cold while the last washes of the membranes are being carried out and put them in a 37ºC water bath so any precipitate can dissolve, and then leave them to come to room temperature; all this normally takes 45 minutes.
Although the instructions recommend the use of a hybridization oven at room temperature for all the incubations and washes, we like to use a rolling-platform which can be easily used at room temperature for that purpose. This allows us the use of the oven at 65ºC for hybridization of more membranes.
In general I like this kit, it’s easy and clean to use, with a good result at the end. The only negative point, in my opinion, is the lack of information about the components of the different buffers, which would be nice to know.