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

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.

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.

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.

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.

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.

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

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. 

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.

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.

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.