BCIP/TNBT Single Reagent, Blue

BCIP/TNBT Single Reagent, Blue/brown, Alkaline Phosphatase Substrate (insoluble)

Chemicon (Millipore)

Although nitroblue tetrazolium with indoxyl phosphate is considered an excellent substrate for alkaline phosphatase immunoblotting procedures, the availability of highly pure tetranitroblue tetrazolium (5-bromo-4-chloro-3-indoxyl phosphate/tetranitroblue tetrazolium) (TNBT) may now allow the immunologist and molecular biologist to achieve even greater sensitivity. Chemicon International has made available a refrigerator-stable, single component BCIP/TNBT solution for Western, Northern, and Southern techniques as well as immunohistochemical and in situ hybridization applications. The reaction product observed is a darker purple than that observed with NBT and may be useful for detection of multiple alkaline phosphatase probes when used in conjunction with BCIP/NBT. Methodology and reaction times are similar to the BCIP/NBT system, but may be shortened because of increased sensitivity.

NOTE: DO NOT USE PHOSPHATE BUFFERS FOR WASHING. Inorganic phosphate is a powerful inhibitor of AP.

• Western Blotting
• Immunohistochemistry

PROCEDURE FOR BCIP/TNBT (ES007):

1) After the final binding reaction with an AP labeled probe, wash tissue sections or membrane thoroughly in a buffer such as Tris/HCl or Tris-buffered saline containing 0.1% Tween 20. DO NOT USE PHOSPHATE BUFFERS. Inorganic phosphate is a powerful inhibitor of AP.

2) After the final wash completely cover tissue sections or membrane with BCIP/TNBT Solution and incubate 5-15 minutes protected from light. Longer incubations may be necessary depending upon enzyme activity. If color develops almost immediately, the formazan deposits may flake off of the tissue sections or membrane. If this occurs, further dilution of the AP probe is necessary. A fine line of formazan deposit circumscribing the band or dot, with no deposit in the center, also suggests further dilution of the AP probes to be necessary.

3) Stop the enzyme reaction by thoroughly washing tissue sections or membranes with deionized water.

4) If desired, counterstain tissue sections in a 0.5% Neutral Red solution prepared in a Mol L(-1) acetate buffer. Hematoxylin solutions will not provide appropriate contrast. Membranes should be dried and stored protected from light. Little or no background staining should be observed on sections or membranes. Presence of excessive background staining indicates incomplete removal of non-bound AP from the sections or membrane. Increase the number of washes or the washing time to remedy this problem.

Results:

A fine dark purple precipitate will localize at sites of AP activity on tissue sections. Dark purple bands or dots will be visible at the sites of AP activity on membranes.

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Black

BCIP/TNBT in a proprietary buffer with enhancer.

Maintain refrigerated at 2-8°C for up to 12 months. Warm to room temperature prior to use. Discard if solution is turbid or purple.

• ELISA Reagents
• Enzyme Substrates

Enzyme Substrates