BCIP/NBT Alkaline Phosphatase Chromogenic Kit

The BCIP/NBT Alkaline Phosphatase Color Development Kit is a versatile tool used for various applications, including immunohistochemistry color development, Western membrane color development, and induced pluripotent stem cell iPS identification. It is composed of a substrate called BCIP/NBT, which is commonly used as a substrate for alkaline phosphatase. This substrate is hydrolyzed by alkaline phosphatase to produce a reactive product that reacts with NBT to form an insoluble, dark blue to blue-purple NBT-formazan.
The kit comes with specific components, including Alkaline Phosphatase Color Buffer, BCIP solution (300x), and NBT solution (150x). The Alkaline Phosphatase Color Buffer has a total volume of 100mL while the BCIP solution and NBT solution are 350µL and 700µL, respectively. These components work together to ensure an accurate and precise color development process.
In summary, the BCIP/NBT Alkaline Phosphatase Color Development Kit is an essential tool used in many biological and biomedical assays. Its high sensitivity and specificity make it a popular choice for researchers and scientists alike.

This substance is commonly used to develop colors in a variety of applications, including immunohistochemistry and Western blotting. It is also helpful for identifying induced pluripotent stem cells (iPS).

To prepare a BCIP solution (100×), it is important to equilibrate it at room temperature for 5 minutes. After equilibration, give it a good shake and thorough mixing. Don't be alarmed if you notice turbidity or precipitation, as this is a normal occurrence due to the high concentration of the BCIP solution. Once the solution is mixed and ready, carefully pipette the desired amount for your experiment.
To prepare the NBT solution, allow it to equilibrate at room temperature for 5 minutes and thoroughly shake and mix it. If turbidity or precipitation occurs, do not worry as this is normal and is caused by the high concentration of BCIP solution. After pipetting and mixing, use the corresponding amount for your experiment with confidence.
After transferring the gel onto the membrane, it is important to wash the membrane with the specified washing solution listed in Table 1 for a duration of 5 minutes.
Table 1 General lotion and blocking liquid
Lotion blocking solution
The washing solution consists of 20 mM Tris-HCl pH 7.5, 0.9% sodium chloride, and 0.5% TWEEN 20.
2 1×TBS + 0.05% TWEEN 20 lotion+1~5% BSA
To make a creamy lotion, simply mix together 1 tablespoon of TBS with 0.05% TWEEN 20 and add in 2-4% skimmed milk powder. This will create a rich and nourishing lotion that will leave your skin feeling soft and smooth. Ensure to mix all the ingredients together thoroughly to create a consistent texture. Enjoy the benefits of this simple yet effective lotion for a healthier, more radiant complexion.
：1；3。33（+2%）..，。
After diluting the primary antibody with blocking solution, incubate with the membrane at room temperature for 2 hours, making sure to shake gently throughout the duration. It is important to dilute the primary antibody according to the instructions provided in its manual.
3. Wash the membrane with lotion for 5 minutes.
4. At room temperature, incubate the membrane with the secondary antibody (secondary antibody conjugated with alkaline phosphatase) diluted with blocking solution for 2 hours, and shake gently. Dilute according to the instructions of the secondary antibody, or dilute the secondary antibody 1000 times with blocking solution before use.
5. Wash the membrane 3 times with lotion for 5 minutes each time.
6. During membrane washing, add each solution in sequence according to the following proportions, and mix it to prepare BCIP/NBT dyeing working solution:
Alkaline Phosphatase Color Buffer 2.95mL 9.833mL
BCIP solution (100×) 30μL 100μL
NBT solution (150×) 20μL 67μL
BCIP/NBT staining working solution 3mL 10mL
7. After the last washing, remove the lotion and add an appropriate amount of BCIP/NBT dyeing working solution to ensure that the sample can be fully covered.
8. Incubate for 1~5min in the dark at room temperature until the color develops to the expected depth.
9. Remove the BCIP/NBT dyeing working solution, wash 1-2 times with distilled water to terminate the color reaction, or rinse with 1% glacial acetic acid to terminate the reaction.

Analysis and solution
1. The background color is too dark
Block before adding the primary antibody. Normal serum (10%) from the same source as the sample, as a secondary antibody, will get the best effect;
The blocking agent added in the western blot, such as 10% BSA, 0.05% TWEEN 20, 3% skimmed milk powder. When using the avidin-biotin system, do not use skimmed milk powder to seal;
Reduce dyeing time;
Use the titration method to determine the optimal dilution factor of the working solution.
2. No color or fuzzy color
Adjust the concentration of the primary antibody;
Adjust the concentration of the secondary antibody;
Determine whether the enzyme coupling is active;
Consider using amplification systems, such as avidin-biotin;
Increase dyeing time;
Adjust the time for transfection of the sample onto the membrane;
Increase sample usage.

1. For your own safety, please protect yourself before using reagents, such as wearing lab coats and wearing gloves.
2. BCIP is irritating to the human body, and NBT is harmful to the human body. Please pay attention to proper protection.
3. Please centrifuge before using BCIP and NBT to avoid losses caused by liquid hanging on the wall.

Store at 2~8° C in the dark for 1 year

BCIP/NBT Alkaline Phosphatase Color Development Kit