BCIP/NBT Stable Liquid Substrate System NBT Enhanced for Alkaline Phosphatase is used for immunoblotting and immunohistochemical procedures. The system is based on hydrolysis of BCIP and reduction of NBT producing a deep purple reaction product.
Single component substrate is more sensitive than existing products and produces little or no background staining.
BCIP/NBT is supplied in an AMP (2-amino-2-methyl-1-propanol) buffer, pH 9.8.
When the phosphate group of BCIP is hydrolyzed by alkaline phosphatase, 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.
When BCIP with Enhanced NBT is added to tissue sections 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 fine purple deposits at the sites of AP activity.
If NBT is present in the system, the reaction proceeds in a different fashion. Reducing components formed during phosphate hydrolysis react rapidly with NBT converting it to the very insoluble purple NBT formazan.
Single component BCIP/NBT reagent is designed for immunohistochemical and in situ hybridization procedures. It may also be used for all blotting methods utilizing AP labeled reagents.
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