Nitrotetrazolium blue chloride for molecular biology protocol

Product Nos. N6639 and N6876

CAS RN 298-83-9
EC 206-067-4
Synonyms: p-Nitro-Blue tetrazolium chloride, p-nitrotetrazolium blue, NBT, Nitro BT

Product Description

NBT is prepared synthetically.1,2 The most common application for NBT is the detection of alkaline phosphatase on western blots.3

NBT has also been used as a redox indicator for other enzymatic reactions including dehydrogenases,4 threonine deaminase,5 glucose-6-phosphate dehydrogenase,6 phosphofructokinase on polyacrylamide gels,7 oxidases on polyacrylamide gels,8 and pentose shunt dehydrogenses.9 Redox and halfwave potentials have been determined for NBT.10,11

NBT has also been used as a colorimetric indicator of bacterial infection in blood samples.12

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices. 

Preparation Instructions

NBT is soluble in H2O at 10 mg/ml, ethanol at 5 mg/ml and 2-methoxyethanol at 20 mg/ml.13 A stock solution at 10 mg/ml in water is stable 1-2 weeks in the dark at 2-8 °C.


NBT has a shelf life of three years when stored at 2-8 °C and protected from light.


Nitro Blue Tetrazolium (NBT) is used with the alkaline phosphatase substrate 5 Bromo-4-Chloro-3-Indolyl Phosphate (BCIP) in immunoblotting5 and immunohistological14 staining procedures. This substrate system produces an insoluble NBT diformazan end product that is blue in color and can be observed visually.

The standard protocol for western blotting is as follows:

  1. Prepare substrate buffer: 0.1 M Tris, 100 mM sodium chloride, 5 mM MgCl2, pH 9.5, adjust pH with HCl.
  2. Prepare NBT stock solution at 10 mg/ml in water.
  3. Prepare BCIP, Product No. B6149, stock solution at 50 mg/ml in water.
  4. Add 33 ml of a 50 mg/ml stock solution of BCIP in water and 330 ml of a 10 mg/ml NBT stock solution in water to 10 ml of substrate buffer.
  5. Rinse specimens incubated with an alkaline phosphatase conjugate in a wash buffer (nonphosphate) before treatment with the BCIP/NBT substrate solution. Cover the entire specimen with the reagent during color development.
  6. Incubate the specimen at room temperature with the BCIP/NBT reagent for approximately 10 minutes. Specimens and procedure may affect the length of time needed for color development.
  7. Monitor color development to avoid overdevelopment. Stop color development by rinsing the specimen with distilled water.

Product Profile

Molecular Formula: C40H30N10O6 · 2Cl
Formula Weight: 817.64
Melting Point: 205 °C1, 189-192 °C2 (decomposes)
Extinction Coefficients:
     260 nm: E1% = 740 (in water)13
     257 nm: Molar Extinction Coefficient = 61,300 (in water)15




  1. Kwan-Chung, T., et al., Syntheses of some pnitrophenyl substituted tetrazolium salts as electron acceptors for the demonstration of dehydrogenases. JACS, 78, 6139-6144 (1956).
  2. Karmarkar, S.S., et al., Synthesis of p-nitrophenyl substituted tetrazolium salts containing iodine and other groups. JACS, 81, 3771 (1959).
  3. Blake, M.S., A rapid, sensitive method for detection of alkaline phosphatase-conjugated antiantibody on Western blots. Anal. Biochem., 136, 175 (1984).
  4. Fine, I.H. and Costello, L.A., The use of starch electrophoresis in dehydrogenase studies. Meth. Enzymol., 6, 958 (1963).
  5. Feldberg, R.S. and Datta, P., Threonine deaminase: a novel activity stain on polyacrylamide gels. Science, 170, 1414-1415 (1970).
  6. Criss, W.E. and McKerns, K.W., Purification and partial characterization of glucose 6-phosphate dehydrogenase from cow adrenal cortex. Biochemistry, 7, 125-134 (1968).
  7. Brock, D.J.H., Purification and properties of sheep liver phosphofructokinase. Biochem. J., 113, 235- 242 (1969).
  8. Feinstein, R.N. and Lindahl, R., Detection of oxidases on polyacrylamide gels. Anal. Biochem., 56, 353-360 (1973).
  9. Altmann, F.P., The use of eight different tetrazolium salts for a quantitative study of pentose shunt dehydrogenation. Histochemie, 19, 363-374 (1969).
  10. Horwitz, J.P., et al., Substrates for Cytochemical demonstration of enzyme activity. II. Some Dihalo-3-indolyl phosphates and sulfates. J. Med. Chem., 9, 447 (1966).
  11. Karmarkar, S.S., et al, Preparation of nitrotetrazolium salts containing benzothiazole. J. Org. Chem., 25, 575-585 (1960).
  12. Bergmeyer, H. U., Methods of Enzymatic Analysis, 3rd ed., 1, 198 (1983).
  13. Green, F.J. (ed.) The Sigma-Aldrich Handbook of Stains, Dyes & Indicators (Aldrich Chemical Co., Milwaukee, WI), p. 523 (1990).
  14. Park, B.H., et al., Infection and nitrobluetetrazolium reduction by neutrophils. A diagnostic acid. The Lancet 2, 532-4 (1968).
  15. Altman, F.P., Tetrazolium salts: a consumer's guide. Histochemical J., 8, 471 (1976).


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