Thermo Scientific™ 1-Step™ NBT/BCIP plus Suppressor Substrate Solution, 100mL

The Thermo Scientific™ Pierce NBT and BCIP Substrates are specialized chemical solutions designed for chromogenic blotting and immunohistochemistry (IHC) applications. These substrates consist of two key components: nitro-blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3'-indolyphosphate (BCIP). These chemicals work in combination to create a visual precipitate that aids in the detection of alkaline phosphatase (alk-phos) activity.

Alkaline phosphatase is a commonly used enzyme conjugate for antibody probes, and the reaction between NBT and BCIP forms an intense, insoluble purple-black precipitate. This precipitate is highly visible and can be used to identify and confirm the presence of the enzyme in various biological samples, such as membrane-bound proteins or fixed tissue specimens.

The Thermo Scientific™ 1-Step™ NBT/BCIP Plus Suppressor Substrate Solution (Catalog number: 34070) includes a levamisole suppressor, which inhibits endogenous phosphatase activity—a common problem in tissue samples that can interfere with the assay results. The inclusion of levamisole ensures a cleaner, more specific signal by preventing the activation of endogenous alkaline phosphatases that could otherwise generate unwanted background noise or false positives in the staining process.

Features of Thermo Scientific™ Pierce NBT and BCIP Substrates

1. Alkaline Phosphatase Substrate:
   • The NBT and BCIP combination is specifically formulated to detect alkaline phosphatase (alk-phos) activity in immunoblotting and immunohistochemistry (IHC) applications. Alkaline phosphatase is a frequently used reporter enzyme conjugated to antibodies, allowing for precise detection of target antigens.
2. Chromogenic Detection:
   • The reaction produces a purple-black precipitate that is visible to the naked eye, making the process accessible without the need for complex imaging equipment. This simplifies the visualization process and allows researchers to quickly confirm results. The colorimetric reaction is a convenient and cost-effective method for protein detection.
3. Ready-to-use Solution:
   • The substrate is available in two main forms: a liquid solution and as powders that can be dissolved to prepare the solution as needed. The ready-to-use solutions ensure consistency and reliability in results, especially when a high degree of sensitivity is required for detection.
4. Choice of Package Options:
   • Users can choose between individual NBT and BCIP powders, or preformulated, ready-to-use solutions. The 1-Step™ NBT/BCIP Plus Suppressor Solution (Part No. 34070) includes a suppressor for reducing endogenous phosphatase interference, ensuring a clearer, more reliable result.
5. Levamisole Suppressor:
   • The 1-Step™ NBT/BCIP Plus Suppressor Solution includes 1 mM levamisole, which specifically inhibits endogenous phosphatases. This helps to reduce background staining and enhances the contrast of the actual target signal in tissue sections, improving the accuracy and sensitivity of the test.

Key Highlights

• Immunoblotting (Western Blotting): Ideal for protein detection applications where alkaline phosphatase is used as a reporter enzyme.
• Immunohistochemistry (IHC): Essential for tissue section analysis, particularly when endogenous phosphatases could cause interference. The suppressor in the solution ensures minimal background signal for more accurate staining.
• High Sensitivity with Low Background: The reaction produces a dark purple precipitate, which is not only easily visible but also highly sensitive with low background noise, enhancing the clarity of results and minimizing assay-to-assay variability.
• Single Component Solution: The ready-to-use solution simplifies the procedure, reducing the chance of error associated with preparing and mixing reagents manually.

Applications

• Western Blotting: Used for protein detection when alkaline phosphatase-conjugated antibodies are used to identify specific proteins separated by gel electrophoresis. The precipitate generated by the NBT/BCIP reaction can be photographed and analyzed.
• Immunohistochemistry (IHC): The NBT/BCIP reaction is used to detect specific proteins or antigens in tissue sections or fixed cells. The 1-Step™ NBT/BCIP Plus Suppressor Solution is especially useful in IHC because it helps reduce the interference caused by endogenous alkaline phosphatases in tissues, ensuring clearer and more reliable staining of the target proteins.
• In Situ Hybridization (ISH): This substrate solution can also be used in various in situ hybridization assays where alkaline phosphatase is used as a detection marker for nucleic acid sequences.

Specifications

Storage and Handling

• Storage: The product should be stored at 4°C upon receipt to maintain its stability and effectiveness. It is shipped with an ice pack to ensure the integrity of the solution during transit.
• Shelf Life: The product has a long shelf life when stored correctly at 4°C. Always check the expiration date before use to ensure maximum performance.

Related Products

• 1-Step™ NBT/BCIP Substrate Solution (without suppressor)
• NBT Substrate Powder (nitro-blue tetrazolium chloride)
• BCIP Substrate Powder (5-bromo-4-chloro-3'-indolyphosphate p-toluidine)

This product is for research use only and not for diagnostic procedures.