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商品详情

Description
OverviewReady-to-use alkaline phosphatase substrate solution. Suitable only for immunoblotting.
Catalogue Number203790
Brand FamilyCalbiochem®
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

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.

Storage and Shipping Information
Ship CodeAmbient Temperature Only
ToxicityStandard Handling
Storage+2°C to +8°C
Protect from LightProtect from light
Do not freezeOk 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.


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