BCIP/NBT Stable Liquid Substrate System for Alkaline Phosphatase has been developed for the localization of AP-labeled probes on Western, Northern, Southern and Dot Blots. The system is based on hydrolysis of BCIP and reduction of NBT producing an intense purple reaction product.
Single component substrate is more sensitive than existing products and produces little or no background staining. BCIP/NBT Stable Liquid Substrate is supplied as a liquid in an AMP (2-amino-2-methyl-1-propanol) buffer, pH 9.8.
When the phosphate group of BCIP is hydrolyzed by alkaline phosphatase (AP) the resulting indolyl derivative adds to itself forming an insoluble, blue halogenated indigo. The reaction is slow and the indigo derivative is slightly soluble in aqueous alkaline buffer solutions. If NBT is present in the system, the reaction proceeds in a different fashion.
When BCIP/NBT solution is added to a blotting system containing alkaline phosphatase labeled probes, hydrolysis of the phosphate ester by AP results in the formation of powerful reducing products. These substances reduce NBT to the insoluble NBT formazan readily visible as purple deposits at the sites of AP activity.
Note: This product is not recommended for immunohistochemical or in situ hybridization techniques. For the latter methodology BCIP/NBT Enhanced is available. See Catalog# B0765.
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