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NBT / BCIP Solution - Ready to Use Alkaline Phosphatase chromogen (ab7468)

Submit a review Q&A (12)References (3)

Overview

  • Product name

    NBT / BCIP Solution - Ready to Use Alkaline Phosphatase chromogen
    See all Alkaline Phosphatase Chromogen reagents

  • Tested applications

    Suitable for: WB, Dot blotmore details

  • General notes

    NBT / BCIP chromogen (ab7468): a sensitive substrate for alkaline phosphatase detection in immunoblotting. Ready to use formulation.

    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)".

    NBT / BCIP Solution - Ready to Use Alkaline Phosphatase chromogen is an artificially manufactured chromogenic substrate for use in sensitive colorimetric assays. Alkaline phosphatase hydrolyzes the BCIP to 5-bromo-4-chloro-3-indole, which is oxidized by oxygen in the atmosphere, yielding a blue dye. In the presence of NBT, an insoluble dark blue precipitate is formed after a reduction reaction instead.

    To use: Wash the membrane after incubation with the alkaline phosphatase conjugate secondary reagent. Apply sufficient substrate to cover the membrane. Color (purple-turquoise, varies with pH) will develop within 30 minutes. Wash, dry membrane and image.

Properties

Applications

The Abpromise guarantee

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
1/1.

Ready to use formulation. 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
1/1.

Ready to use formulation.
Notes
WB
1/1.

Ready to use formulation. 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
1/1.

Ready to use formulation.

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

References (3)

Publishing research using ab7468? Please let us know so that we can cite the reference in this datasheet.

ab7468 has been referenced in 3 publications.

  • Gigout A  et al. Early detection of osteoarthritis in the rat with an antibody specific to type II collagen modified by reactive oxygen species. Arthritis Res Ther 23:113 (2021). PubMed: 33853645
  • 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). 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 . PubMed: 27406241

Customer reviews and Q&As

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1-10 of 12 Abreviews or Q&A

Question

Dear Technical Team

Our customer boutht ab7468, BCIP/NBP ready to use.
She will use as a westerblot substrate.
Could let me know specific protocol?
For ex, Does she have to dilute?
How long she need incubation time. How to wash.

I am looking forward to your reply,

Best regards

Read More
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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Question

Inquiry: I am looking to use this kit with an AP-conjugated secondary. We currently use HRP secondaries in a SynGene dark-box to detect the chemiluminescence. Would this product be suitable for detection in the same dark-box as there is an option to have an upper white light? Many thanks.

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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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Question

Thanks for your advice and suggestions last time. I got another ab7468 and it is working well, both for WB and my reattempt ELISA.   However I have a question which I hope you may have an answer to:   After adding the pNPP substrate, I waited for 30 min, as recommended, but the signal was very weak. OD was around 0.1 and negative control 0.06. However as I waited for longer, up to 1.5 hour, the signal intensified in the samples and went up to about 0.4. Negative control still remained low at 0.06, which was good. The resultant standard curve was great (R2=0.99). However I could not understand why it took almost 60-90 min before the OD got up to a “good signal”. I don’t think I am simply increasing background as the standard curve was good and the longer duration did not intensify signal from any of the negative controls.   Is this longer than usual duration due to 1)      Inadequate primary ab? 2)      Inadequate secondary ab? 3)      Inadequate coating antigen on plate??  

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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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Question

Do I need to add a stop solution (ie NaOH) at the end of the sandwich ELISA after the colour develops with ALP chromogen or that is not necessary?

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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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Question

I recently purchased your ab (ie Rabbit anti-human FNDC5 IgG), to be used in a ELISA. I am wondering what detection system would you recommend?   Ie According to the Abcam ELISA protocol, one option is HRP.   So Do I add a secondary ab conjugated (ie anti-rabbit IgG Fc, HRP conjugated), followed by washing and then dispensing 100 ul of substrate solution (ie TMB?)   Or would you recommend other secondary/substrate solution system?  

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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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Question

Please advise on the difference between items 1 and 2. 1.            1.     Alkaline Phosphatase chromogen (BCIP/TNBT) (ab7413) 2.     Alkaline Phosphatase chromogen (BCIP/NBT) - Ready to Use (ab7468)  

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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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Question

I am staining 5uM bone sections for endogenous alkaline phosphatase activity. I have applied the solution to the slides but there is no reaction after 1.5 hours. How long would you recommend incubating this? Also, what are the concentrations of BCIP/NBT in this solution? I am concerned that it is not strong enough.

Read More
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

How to resolubilize the chromogen to quantify after the staining?

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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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Question

Hello,I want to buy substrats for Alkaline phosphatase and HRP for double elispot test.I have seen that you have for alkaline phosphatase  BCIP /NBT :ab7413ab7468for HRP AEC :ab103742ab64252but it is only mentionned for IHC.Can I use these reagents for elispot?Do they need to be filtered or not?What is the protocol (volume to dispense, dilution...?)Thanks for your answer.

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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

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