BCIP/NBT 1 Component Membrane Alkaline Phosphatase Substrate

BCIP/NBT 1 Component Membrane Alkaline Phosphatase Substrate

Product No.: B390

[product_table name="All Top" skus="B390"]

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Product Type
Substrate and Assay Reagents
Applications
WB

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Product Size
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Product Details

Storage and Handling
Store at temperatures from 2-25ºC. Do Not Freeze
Expiration Date
Stable for a minimum of 3 years from the manufactured date when properly stored between 2-25ºC. The substrate should not be frozen and should be protected from direct light by storing in amber bottles. Only high quality amber glass and plastic products should be used for storing aliquots.

Description

Background
This BCIP/NBT 1 component buffered alkaline phosphatase substrate (5-bromo-4-chloro-3-indolyl-phosphate and nitroblue tetrazolium) is a precipitating substrate used with the enzyme alkaline phosphatase designed for various membrane immunoassays but not recommended for microwell or immunohistochemical applications. Initially, the unreacted substrate should be colorless to light yellow in appearance. When this substrate system is reacted with alkaline phosphatase, an insoluble, permanent dark purple reaction product is obtained.

Leinco Technologies’ BCIP/NBT 1 Component Membrane Substrate is a highly sensitive, high performance formulation that produces a high signal to noise ratio with low membrane background. The outstanding shelf life of at least thirty six months for the BCIP/NBT 1 Component Membrane Substrate makes this reagent ideal for long term use of the same manufacturing lot.
Directions for Use
BCIP/NBT 1 Component Membrane Substrate is a ready to use solution that needs no preparation or dilution. This product should be at room temperature (25°C) before use. Pour the necessary amount of substrate onto the blocked alkaline phosphatase conjugate labeled membrane to completely cover the membrane. The substrate will react with sites on the membrane containing phosphatase, producing an insoluble purple reaction product. The reaction should be monitored and read before the background color becomes too intense which may result in diminished contrast between the positive and background staining. To stop the reaction, rinse membrane with reagent quality water. Some wash solutions containing detergents may reduce color intensities. Dilution of the substrate is not recommended. To reduce the intensity of a reaction, it is recommended that the antibodies or conjugates be diluted.
Troubleshooting
 
Problem Cause Solution
Too much background signal observed · BCIP/NBT substrate was left on the membrane too long · Decrease the amount of time the BCIP/NBT substrate is on the membrane
· Too much primary antibody used · Decrease the amount of primary antibody used and wash the membrane sufficiently after the primary antibody incubation
· Too much secondary antibody used · Decrease the amount of secondary antibody used
Nonspecific bands show up on the membrane · Too much primary antibody used · Decrease the amount of primary antibody used and wash the membrane sufficiently after the primary antibody incubation
· Too much secondary antibody used · Decrease the amount of secondary antibody used
Signal disappears from membrane · Membrane not stored correctly · Store the membrane dry in plastic bag in the dark
No signal is observed on the membrane · Low amounts of specific signal present · Expose the membrane to BCIP/NBT substrate for a longer period of time. Include positive control(s) during analysis
· Insufficient primary antibody used · Use more primary antibody
· Insufficient secondary antibody used · Use more secondary antibody
· Sample degraded · Add protease inhibitors to original sample before running a gel

Related Protocols

General Western Blot Protocol
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Products are for research use only. Not for use in diagnostic or therapeutic procedures.