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Preparation of BCIP/NBT Color Development Solution

2018-03-13 来源:亚科官网

BCIP/NBT is one of the substrate combinations for alkaline phosphatase (AP) and is used to detect antigen bound to nitrocellulose or other membranes. Under the catalysis of AP, BCIP is hydrolyzed to produce a strong reactive product that reacts with NBT to form an insoluble dark blue to bluish-purple product. The corresponding kit can be used for enzymatic chromogenic reaction of the alkaline phosphatase system in IHC and WB experiments. Under the catalysis of AP, a deep blue precipitate is generated at the place where the AP conjugate is bound on the tissue section or the blot membrane, and the position and expression of the target protein can be determined according to the color reaction. The following briefly describes the preparation method of the BCIP/NBT coloring solution:

Preparation of BCIP, NBT Solution and Buffer

(1) 1L Tris buffer (0.1 M Tris, 0.5 mM magnesium chloride, pH=9.5)

  12.1 g of Tris and 0.12 g of magnesium chloride hexahydrate were added to 800 ml of distilled and deionized water (dd H2O). Then, the pH was adjusted to 9.5 with hydrochloric acid, and finally dd H2O was added to obtain 1 L of Tris buffer.

(2) NBT solution

   0.7 ml of DMF and 0.3 ml of dd H2O were mixed to give 1 ml of a solution containing 70% DMF, and then 30 mg of NBT was dissolved therein.

(3) BCIP solution

   Add 15 mg of BCIP to 1 ml of DMF to obtain a BCIP solution.

Preparation of BCIP/NBT color solution

 The above-prepared NBT solution and the BCIP solution were mixed and added to 100 ml Tris buffer to obtain a corresponding color development solution.


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