Millipore Sigma Vibrant Logo
Attention: We have moved. Merck Millipore products are no longer available for purchase on MerckMillipore.com.Learn More

203790 BCIP/NBT

BCIP/NBT MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

Synonyms: 5-Bromo-4-chloro-3-indolylphosphate/Nitroblue Tetrazolium

203790
Purchase on Sigma-Aldrich

Overview

Replacement Information

Products

Catalogue NumberPackaging Qty/Pack
203790-100ML Glass bottle 100 ml
Description
OverviewReady-to-use alkaline phosphatase substrate solution. Suitable only for immunoblotting.
Catalogue Number203790
Brand Family Calbiochem®
Synonyms5-Bromo-4-chloro-3-indolylphosphate/Nitroblue Tetrazolium
References
ReferencesDe Jong, A.S., et al. 1985. Histochem. J. 17, 1119.
Product Information
FormClear to pale yellow liquid
Formulation0.577 mM BCIP, 0.122 mM NBT in a proprietary solution.
PreservativeNone
Quality LevelMQ100
Applications
Application NotesImmunoblotting (see comments)
Application CommentsIt is not recommended for immunohistochemistry or in situ hybridization. Functionality check performed on final product by dot blot. Additional QC testing is preformed on a lot-specific basis:
pH: 9.7-9.9
Stability at 4°C
Stability at 18-26°C

Suggested procedure for immunoblot staining with BCIP/NBT:

1. Complete all required incubations with antibodies and alkaline-phosphatase-labeled probes.
2. After the final binding reaction with the alkaline-phosphatase labeled probe, wash the membrane thoroughly in a buffer such as Tris-buffered saline (TBS) containing 0.1% Tween®-20 detergent. DO NOT USE PHOSPHATE BUFFERS; inorganic phosphate is a potent inhibitor of alkaline phosphatase.
3. Following the final wash, completely cover the membrane with BCIP/NBT Solution and incubate, protected from light, for 5-15 min, or until the desired color intensity is obtained (dark purple bands or dots will appear at the sites of enzyme activity).
Note: Variables associated with assay conditions will dictate the proper reaction time. If the color develops almost immediately, the NBT-formazan deposit may flake off the membrane. If this occurs, further dilution of the alkaline phosphatase probe is recommended. A fine line of formazan deposit circumscribing the band or dot, with no deposit in the center, also suggests that further dilution of the alkaline phosphatase probe is needed.
4. Stop the reaction by washing the membrane thoroughly in distilled water.
Note: There should be little or no background. Presence of excessive background staining indicates incomplete removal of unbound alkaline phosphatase from the membrane. To remedy this problem, increase the number of wash steps or washing times.
5. Air-dry membranes and store at room temperature, protected from light.
Biological Information
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Product Usage Statements
Storage and Shipping Information
Ship Code Ambient Temperature Only
Toxicity Standard Handling
Storage +2°C to +8°C
Protect from Light Protect from light
Do not freeze Ok to freeze
Special InstructionsDiscard if the solution turns purple or turbid. The reagent is stable at room temperature, but we recommend storage in the refrigerator to increase the shelf life. Warm to assay temperature before use.
Packaging Information
Transport Information
Supplemental Information
Specifications

Documentation

BCIP/NBT SDS

Title

Safety Data Sheet (SDS) 

BCIP/NBT Certificates of Analysis

TitleLot Number
203790

References

Reference overview
De Jong, A.S., et al. 1985. Histochem. J. 17, 1119.
Data Sheet

Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.

Revision20-May-2008 RFH
Synonyms5-Bromo-4-chloro-3-indolylphosphate/Nitroblue Tetrazolium
ApplicationImmunoblotting (see comments)
DescriptionBCIP/NBT provided as a stable single component, ready-to-use substrate, sterile-filtered through a 0.22 µm filter. Specifically designed for immunoblotting techniques.
BCIP/NBT is the substrate of choice for alkaline phosphatase-based immunoblotting procedures. Hydrolysis of the phosphate ester of BCIP by alkaline phosphatase yields powerful reducing compounds that react rapidly with NBT and convert it to the insoluble NBT-formazan. This reaction product is readily visible on the blotting membrane as dark purple deposits that do not fade upon drying.
BackgroundBCIP/NBT is the substrate of choice for alkaline phosphatase-based immunoblotting procedures. Hydrolysis of the phosphate ester of BCIP by alkaline phosphatase yields powerful reducing compounds that react rapidly with NBT and convert it to the insoluble NBT-formazan. This reaction product is readily visible on the blotting membrane as dark purple deposits that do not fade upon drying.
FormClear to pale yellow liquid
Formulation0.577 mM BCIP, 0.122 mM NBT in a proprietary solution.
Recommended reaction conditionsSuggested procedure for immunoblot staining with BCIP/NBT: 1. Complete all required incubations with antibodies and alkaline-phosphatase-labeled probes. 2. After the final binding reaction with the alkaline-phosphatase labeled probe, wash the membrane thoroughly in a buffer such as Tris-buffered saline (TBS) containing 0.1% Tween®-20 detergent. DO NOT USE PHOSPHATE BUFFERS; inorganic phosphate is a potent inhibitor of alkaline phosphatase. 3. Following the final wash, completely cover the membrane with BCIP/NBT Solution and incubate, protected from light, for 5-15 min, or until the desired color intensity is obtained (dark purple bands or dots will appear at the sites of enzyme activity). Note: Variables associated with assay conditions will dictate the proper reaction time. If the color develops almost immediately, the NBT-formazan deposit may flake off the membrane. If this occurs, further dilution of the alkaline phosphatase probe is recommended. A fine line of formazan deposit circumscribing the band or dot, with no deposit in the center, also suggests that further dilution of the alkaline phosphatase probe is needed. 4. Stop the reaction by washing the membrane thoroughly in distilled water. Note: There should be little or no background. Presence of excessive background staining indicates incomplete removal of unbound alkaline phosphatase from the membrane. To remedy this problem, increase the number of wash steps or washing times. 5. Air-dry membranes and store at room temperature, protected from light.
PreservativeNone
CommentsIt is not recommended for immunohistochemistry or in situ hybridization. Functionality check performed on final product by dot blot. Additional QC testing is preformed on a lot-specific basis:
pH: 9.7-9.9
Stability at 4°C
Stability at 18-26°C

Suggested procedure for immunoblot staining with BCIP/NBT:

1. Complete all required incubations with antibodies and alkaline-phosphatase-labeled probes.
2. After the final binding reaction with the alkaline-phosphatase labeled probe, wash the membrane thoroughly in a buffer such as Tris-buffered saline (TBS) containing 0.1% Tween®-20 detergent. DO NOT USE PHOSPHATE BUFFERS; inorganic phosphate is a potent inhibitor of alkaline phosphatase.
3. Following the final wash, completely cover the membrane with BCIP/NBT Solution and incubate, protected from light, for 5-15 min, or until the desired color intensity is obtained (dark purple bands or dots will appear at the sites of enzyme activity).
Note: Variables associated with assay conditions will dictate the proper reaction time. If the color develops almost immediately, the NBT-formazan deposit may flake off the membrane. If this occurs, further dilution of the alkaline phosphatase probe is recommended. A fine line of formazan deposit circumscribing the band or dot, with no deposit in the center, also suggests that further dilution of the alkaline phosphatase probe is needed.
4. Stop the reaction by washing the membrane thoroughly in distilled water.
Note: There should be little or no background. Presence of excessive background staining indicates incomplete removal of unbound alkaline phosphatase from the membrane. To remedy this problem, increase the number of wash steps or washing times.
5. Air-dry membranes and store at room temperature, protected from light.
Storage Protect from light
+2°C to +8°C
Do Not Freeze Ok to freeze
Special InstructionsDiscard if the solution turns purple or turbid. The reagent is stable at room temperature, but we recommend storage in the refrigerator to increase the shelf life. Warm to assay temperature before use.
Toxicity Standard Handling
ReferencesDe Jong, A.S., et al. 1985. Histochem. J. 17, 1119.