Overview

  • Product name
    Alkaline Phosphatase chromogen (BCIP/TNBT)
    See all Alkaline Phosphatase Chromogen reagents
  • Tested applications
    Suitable for: WB, IHC-P, IHC-Frmore details
  • General notes

    This product is a liquid substrate for alkaline phosphatase (AP), and is especially useful for the detection of AP-conjugated antibodies (i.e. secondary antibodies). Apply to tissue section for 10 minutes or until a suitable level of colouration has been achieved.

    BCIP: 5-bromo-4-chloro-3-indolyl phosphate 0.452 mM TNBT: tetranitroblue tetrazolium 0.432 mM This product used to be called "Alkaline phosphatase detection kit (BCIP/TNBT)".

    BCIP/TNBT is similar to BCIP/NBT but is more sensitive and produces a more intense purple color. A useful secondary marker in multiple staining procedures.

Properties

Applications

Our Abpromise guarantee covers the use of ab7413 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.
IHC-P Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.

References

ab7413 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

Question
Answer

Thank you for your enquiry.

I am sorry to confirm that ab133406 is not suitable for detection of AP. This detection system has been designed for optimal detection of HRP, and will not be as sensitive for detection of AP.

I am sorry I am not sure which application you are using, but the following two detection substrate kits are suitable for western blotting, and should also be suitable for IHC:

ab7413 Alkaline Phosphatase chromogen (BCIP/TNBT) https://www.abcam.com/index.html?datasheet=7413 https://www.abcam.com/index.html?datasheet=7413.

ab7468 Alkaline Phosphatase chromogen (BCIP/NBT) https://www.abcam.com/index.html?datasheet=7468 https://www.abcam.com/index.html?datasheet=7468.

I can recommend to review the online datasheets carefully for further information.

I hope this information will be helpful to you. If you have any further questions, please do not hesitate to contact us.

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Answer

Thank you for contacting us.

As requested, I have attached the Certificates of Compliance for the antibodies and reagents you mentioned - to this email.

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

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you for confirming the order number.
This is to let you know that I have confirmed and agreed on placing a new order for your customer - for one vial of ab64268 (new and different Batch:GR71729-1) as a free of charge replacement. I hope the second vial will work as it is expected, and please do let me know how you are getting on with this product.
Dear Colleague,
Please add 1 vial of ab64268 (Batch:GR71729-1) to the next shipment for Sapphire Bioscience Pty Ltd.
Original CCE ID: 3445319
Original Order: 948346
Problem is: ALP is inactive and no signal in IHC with this reagent
Many thanks.

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Answer

Thank you for getting back to me and for re-testing the ALP (AP) activity with new and old chromogen solution. Your co-operation and understanding in this matter is very much appreciated!
I can certainly replace the faulty product (ab64268) as soon as possible.
Once again I apologize for any inconvenience caused and thank you for bringing this matter to our attention.

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Answer

Thank you very much for getting back to me promptly and for checking the ALP (AP) activity.
This is very important piece of detail and it may indeed imply that AP is not working properly. As I understand Streptavidin Alkaline Phosphatase (ab64268) was purchased from Abcam via the Distributor as well. Would you be so kind to confirm the batch number and the order number of this item as well?
Could you please let Sapphire and myself know how you wish to proceed with this enquiry?
I look forward to hearing from you soon.

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Answer

Thank you very much for confirming the order details.
Could you please comment on if the ALP (AP) enzyme conjugated to the antibody is inactive? Have you had any chance to check if the ALP is still functioning?
Would you prefer to get a new product or a credit note?
I look forward to hearing from you soon.

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Question

LOT NUMBER GR7874-3 ORDER NUMBER 0761003659 UWA DESCRIPTION OF THE PROBLEM Reaction product is not developing. I am using the solution neat at room temp incubating for up to 30minutes. I have compared the same primaries on the same tissue using immunofluorescence, DAB staining and the AP staining using components bought on the same order (biotinylated anti mouse, biotinylated ant rabbit, streptavidin AP. Staining is positive in fluorescence, and DAB developed sections. Also I have tried the AP BCIP/TNBT without success then applied streptavidin HRP and developed with DAB and got postiive although weaker staining showing that the problem seems to lie with the enzymatic step. Are there any other recommendations? Has anyone else had problems with this reagent? The dilution of the antibody doesn't seem to be a problem, nor do these antibodies usually require antigen retrieval. SAMPLE Rat optic nerve, transverse sections 14 microns, cryosectioned. PRIMARY ANTIBODY Abcam Ab 18207 rabbit B3 Tubulin Covance mouse B3 Tubulin Dako rabbit GFAP DETECTION METHOD Abcam Ab 7413 - not working DAB kit using Streptavidin HRP and developing with DAB and substrate buffer - working Immunofluorescence - working POSITIVE AND NEGATIVE CONTROLS USED Positive controls - same tissue, antibody dilution, incubation times for fluorescence and DAB staning. ANTIBODY STORAGE CONDITIONS 4 deg C in original bottle FIXATION OF SAMPLE 4% PFA 1-2 days, 15% sucrose overnight, cryosectioned at 14microns ANTIGEN RETRIEVAL Not required for this tissue/fix/antibody combination PERMEABILIZATION STEP 0.2% triton in PBS BLOCKING CONDITIONS 5% Normal Goat serum but no blocking also been tried (no difference in immunofluorescence). SECONDARY ANTIBODY Abcam ab 64256 biotinylated goat anti rabbit Abcam Ab 64255 biotinylated goat anti mouse Abcam Ab 64268 Streptavidin AP Fluorescence- Alexafluor anti mouse or rabbit 488 or 555 HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? I have tried a different Strep AP (old stock) I extended the incubation time for the enzyme reaction. DAB develops within less than 5 mins, so I gave AP 10-15 mins first time then extended to 30 mins.

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Answer

Thank you for your enquiry regarding ab7413 and for taking the time to provide some very useful details of the experiments. Your time and effort is much appreciated!
I am very sorry to hear that you are having problems with this chromogen.
As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.
It could well be that this product has been damaged accidentally either during shipment or storage.
The other alternative would be that the ALP (AP) enzyme conjugated to the antibody is inactive. Have you had any chance to check if the ALP is still functioning?
Would you be so kind to kind the Abcam Order and the date of purchase, our system does not recognize 0761003659 UWA. You may need to contact Sapphire to get the requested information.
I look forward to hearing from you and hope to solve this problem as soon as possible.

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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 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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Answer

This substrate would detect any endogenous alkaline phosphatase if it were enzymatically active, but I am not sure how activity could remain since alkaline phosphatase is normally inactivated by freezing and chemical treatment during fixation.

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

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