Overview

  • Product name
    Alkaline Phosphatase chromogen (BCIP/NBT) - Ready to Use
    See all Alkaline Phosphatase Chromogen reagents
  • Tested applications
    Suitable for: WB, Dot blot, IHC-P, IHC-Frmore details
  • General notes
    BCIP: 5-bromo-4-chloro-3-indolyl phosphate NBT: p-nitroblue tetrazolium chloride This product used to be called "Alkaline phosphatase detection kit (BCIP/NBT)". Abcam has a wide range of primary and secondary antibodies available on the Abcam website to suit your protocol needs.

Properties

Applications

Our Abpromise guarantee covers the use of ab7468 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB Use at an assay dependent concentration.
BCIP/NBT system is based on hydrolysis of BCIP and reduction of NBT producing a deep purple reaction product. Reaction is several times more sensitive than existing products with little or no background staining. The reaction product is extremely stable and does not fade when exposed to light.
Dot blot Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.

References

This product has been referenced in:
  • Hassani A  et al. Epstein-Barr virus is present in the brain of most cases of multiple sclerosis and may engage more than just B cells. PLoS One 13:e0192109 (2018). Read more (PubMed: 29394264) »
  • Song JW  et al. 3'-UTR engineering to improve soluble expression and fine-tuning of activity of cascade enzymes in Escherichia coli. Sci Rep 6:29406 (2016). WB . Read more (PubMed: 27406241) »
See all 2 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Answer

Thank you for your inquiry.

I can confirm that the ab7468 Alkaline Phosphatase chromogen (BCIP/NBT)substrate is ready to use:

Wash the membrane after incubation with the alkaline phosphatase secondary.
Apply sufficient substrate to cover the membrane.
Color (purple-turquise, varies with pH) will develop within 30 minutes.
Rinse, dry and image.

I hope this will be helpful. Please feel free to contact me if you have more questions.

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Answer

Thank you for contacting us.

NBT/BCIP system produces a colorimetric substrate that yields an intense and insoluble black-purple precipitate when reaction with alkaline phosphatase.

Unlike HRP chemiluminiscent methods, the chromogenic product produced by the reaction of NBT/BCIP with AP does not require any special equipment for visualization.

I hope this helps. Please let me know if you require any further assistance.

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Question

Many thanks for your reply. I really appreciate your suggestions. My detection is working well now. However I am facing some strange results which I would love your thoughts on please:   I am struggling to interpret an indirect ELISA I am making. It involved coating wells with human plasma overnight, blocked with milk the next day, then primary ab (Abcam), then detection ab (Abcam) and finally the ALP substrate. I ran parallel wells in which I coated with   1)      known concentration of antigen which is a recombinant protein to generate my standard curve and 2)      BSA (negative control) I got nice reproducible standard curves (R2=0.99) and uniformly negative signals from BSA. So all good. However I am seeing a dose-dependent increase in signal in plasma samples. As plasma dilution increased, signal increased - in other words, the more dilute the plasma (so in theory lesser amount of antigen) - the stronger the signal! I could not understand why. I repeated the experiments and I am seeing it every time (4x now). At first I thought it could be due to primary ab binding to "unoccupied sites in the wells" directly, which then generated a signal when secondary added, which would explain why the signal is stronger in diluted sample. But if that's the case I should see that in the BSA wells, but I am not. I did block the wells with milk after coating to ensure all unbound sites are occupied. Then I thought could it be something in the plasma which is inhibiting the substrate reaction (ALP). So the more dilute the plasma, the less the inhibition, and the stronger the signal. But plasma was only used to coat the plate overnight. The plates were then washed after every step with TBS-tween (2-4 times) after every step. So I would imagine not much of any “inhibitors” are around by the time substrate is added. Then I thought whether it could be a “hook” effect. In other words, the plasma is too concentrated and antigen are crowding up thus reducing the amount of binding. But I thought usually plasma then need to be diluted up to 100 folds to relieve the hook. But I am seeing a “nice” dose dependent increase in signal as plasma was diluted from 1:2, 1:4, 1:8 to 1:16 only.

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Answer

Thank you for contacting me again. It is always a pleasure to hear from you.

After reviewing your email, I am lead to believe that this really is a hook effect as you had mentioned. I am not an expert on this hook effect in plasma specifically, and so cannot comment on the 100 fold dilutions usually required, but your description does sound like a hook effect. These should eventually tail off with further dilutions.

The hook effect may also be created by overcoating with primary antibody; you may wish to further dilute your primary to tackle this as well.

I hope these suggestion help. Let me know if you have any questions.

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Answer

Thank you for contacting us, I am glad to hear that the new product is working well for you.

Usually when color is developing slowly it is one of three issues. 1) the plates and reagents have not come to room temperature. 2) the conjugates are weak, try to prepare these right before use and at correct concentration 3) the presence of peroxidases, azide or other contaminants are interfering with the reaction, these may be removed by dyalisis, gel filtration or with purification columns.

I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

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Answer

Thank you for contacting us.

Just as with a common indirect ELISA it is important to stop the reaction using the appropriate stop solution.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting Abcam.

Enzymatic detection with an antibody that has been conjugated to HRP or to AP is a widely used and robust method. As this target is located on the peroxisome membrane I would recommend using anAlkaline Phosphataseconjugated secondary for this target prehaps ab6722,Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (AP). Followed by an appropriate substrate detection of your choice. I recommend theAlkaline Phosphatase chromogen (BCIP/NBT). We do offer this as a ready to use product, ab7468.

I hope you have found this information helpful. Please let me know if you have any questions. I have places links to each of the products I have mentioned here below.

Ab6722:https://www.abcam.com/Goat-polyclonal-Secondary-Antibody-to-Rabbit-IgG-H-L-AP-ab6722.html

Ab7468:https://www.abcam.com/Alkaline-Phosp

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Answer

Thank you for contacting us.  BCIP/TNBT (ab7413) is similar to BCIP/NBT (ab7468) but is more sensitive and produces a more intense purple color.  Otherwise, the two products will be interchangeable.  I hope this helps, please let me know if you need any additional information. 

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Answer

Thank you for contacting Abcam regarding ab7468. I contacted the laboratory for the BCIP/NBT concentration, however this information appears to be proprietary. A signal is typically expected within 20 minutes, however we have not specifically tested for endogenous alkaline phosphatase activity with this kit. It is possible that a longer incubation is necessary to detect a signal. Please let me know if the longer incubation improves your signal. Also, I would recommend adding some diluted secondary antibody to the solution to see if it turns blue/purple. Do not hesitate to contact me if you have any additional questions.

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Question
Answer

Thank you for your inquiry. I have contacted the laboratory, however they do not have a protocol for resolubilizing this chromagen. I tried to search the internet, could however not find an adapted protocol for this. I am very sorry for not being able to help you more on this occasion. I hope you will find a solution to this problem. Please do not hesitate to call us back if you any other question or concern.

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Answer

Thank you very much for your enquiry. We do not currently sell substrates such as AP and HRP that have been tested in ELISPOT. This doesn’t mean that these products are not suitable, we just have not tested them in this application. The 4 products identified (ab7413, ab7468, ab64252, ab103742) have been tested in IHC and ab7413 has also been tested in dot blots and WB. This may be a more suitable one to try but unfortunately we cannot guarantee that it would be suitable. I would consider reviewing what ELISPOT kits we have available as we may have one that is suitable. I would be happy to offer any further advice and hope this information helps.

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1-10 of 12 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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