Product overview

Name NBT/BCIP Stock Solution
Biological description

Overview

Suitable as substrate for alkaline phosphatase detection in applications such as IHC & immunoblotting (e.g. Dot blot). Produces an insoluble, blue- purple end product following reaction with alkaline phosphatase (AP).

Contents

Solution of 18.8 mg/ml nitro-blue tetrazolium chloride; 9.4 mg/ml 5-bromo-4-chloro-3-indolylphosphate toluidine salt in 67% DMSO.

Preparation note - preparation of staining solution

Always prepare fresh solutions, shortly before use.

  • For all applications except DIG System: Add 200 μl of the stock solution to 10 ml 0.1 M Tris-HCl, pH 9.5 (20 °C), 0.1 M NaCl, 0.05 M MgCl2.
  • For DIG System applications: Add 200 μl of the stock solution to 10 ml 0.1 M Tris-HCl, pH 9.5 (20 °C), 0.1 M NaCl

Note: Please do not include MgCl2 in the detection buffer as this can lead to spotty background on the membrane after the detection procedure. Insoluble in water.

Description Alkaline phosphatase detection substrate
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Images

Figure 1. Histoblot for NeuN in rat brain using NBT/BCIP for detection.

Figure 1. Histoblot for NeuN in rat brain using NBT/BCIP for detection. NBT/BCIP is a substrate for alkaline phosphatase conjugated antibodies producing a purple precipitate enabling the visualisation of targeted proteins. NBT/BCIP from Hello Bio enables the visualisation of neurones in rat brain using histoblotting. For more details please see #Protocol 1.

Biological Data

Application notes

#Protocol 1: NeuN Histoblot in rat brain tissue and development using NBT/BCIP

 

  • 10µm fresh frozen horizontal sections were cut from rat brains.
  • Proteins were transferred to nitrocellulose membranes and were probed with HB6429 (mouse monoclonal anti-NeuN antibody) following the protocol detailed in Molnár, 2016. Neuromethods
  • Detection was achieved by diluting the NBT/BCIP stock solution 1:50 into 0.1M Tris-HCl (pH9.5), 0.1M NaCl, 5mM MgCl2  and incubating the solution with the membranes for approximately 30 minutes.
  • To stop the reaction membranes were incubated in PBS.

Solubility & Handling

Storage instructions +4°C
Important This product is for RESEARCH USE ONLY and is not intended for therapeutic or diagnostic use. Not for human or veterinary use

Calculators

Molarity

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Dilution

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Chemical Data

Chemical name 5-Bromo-4-chloro-3-indolyl phosphate disodium salt and 2,2-bis(4-Nitrophenyl)-5,5-diphenyl-3,3-(3,3-dimethoxy-4,4-diphenylene)ditetrazolium chloride
Chemical structure  Chemical Structure
PubChem identifier 71312258
SMILES CC1=CC=C(C=C1)N.COC1=C(C=CC(=C1)C2=CC(=C(C=C2)[N+]3=NC(=NN3C4=CC=C(C=C4)[N+](=O)[O-])C5=CC=CC=C5)OC)[N+]6=NC(=NN6C7=CC=C(C=C7)[N+](=O)[O-])C8=CC=CC=C8.C1=CC(=C(C2=C1NC=C2OP(=O)(O)O)Cl)Br.[Cl-].[Cl-]

References for NBT/BCIP Stock Solution

References are publications that support the biological activity of the product
  • Fluorescent in situ hybridization employing the conventional NBT/BCIP chromogenic stain

    Trinh le A et al (2007) Biotechniques 42(6) : 756-9
  • A high-resolution, fluorescence-based method for localization of endogenous alkaline phosphatase activity

    Cox WG et al (1999) J Histochem Cytochem 47(11) : 1443-56
  • A multiple-staining procedure for the detection of different DNA fragments on a single blot

    West S et al (1990) Anal Biochem 190(2) : 254-8

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