Endogenous Avidin + Biotin Blocking System (ab3387)

Submit a review Q&A (10)References (3)

Overview

  • Product name
    Endogenous Avidin + Biotin Blocking System
    See all Endogenous Avidin+Biotin Blocking System kits
  • Product overview

    Endogenous Avidin + Biotin Blocking System ab3387 is used to decrease the signal caused by endogenous avidin, biotin, or biotin-binding proteins when immunostaining specimens with detection systems that rely on avidin-biotin / steptavidin-biotin (ABC systems). Non-specific staining due to these factors commonly resembles a diffuse, weak, cytoplasmic pattern.

  • Tested applications
    Suitable for: IHC-Fr, IHC-P, ICC/IFmore details

Properties

  • Storage instructions
    Store at +4°C. Please refer to protocols.
  • Storage buffer
    Preservative: 0.1% Sodium Azide in both components
  • Components 1 kit
    Avidin solution 1 x 15ml
    Biotin Solution 1 x 15ml
  • Research areas
  • Relevance
    Some cells, and tissues such as kidney, liver and spleen, contain endogenous biotin. Using an avidin-biotin staining method may result in high, non-specific background staining. A significant reduction of this non-specific background can be obtained by pre-treatment of cells/tissues with avidin/biotin blocking reagents prior to the incubation of biotinylated antibody.

Applications

Our Abpromise guarantee covers the use of ab3387 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
IHC-Fr Use at an assay dependent dilution.
IHC-P Use at an assay dependent dilution.
ICC/IF Use at an assay dependent dilution.

References

This product has been referenced in:
  • Liakhovitskaia A  et al. Runx1 is required for progression of CD41+ embryonic precursors into HSCs but not prior to this. Development 141:3319-23 (2014). IHC-Fr ; Mouse . Read more (PubMed: 25139854) »
  • Vesterdal LK  et al. Accumulation of lipids and oxidatively damaged DNA in hepatocytes exposed to particles. Toxicol Appl Pharmacol N/A:N/A (2013). Read more (PubMed: 24121055) »
See all 3 Publications for this product

Publishing research using ab3387? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-10 of 10 Abreviews or Q&A

Question

Inquiry: I am trying to do Biotinylation - internalization experiment in which I biotinylate at 4°C, then allow trafficking for different time points. Unfortunately the cell line I am using (T47-D breast cancer cell line) seems to express endogenous biotin which affect the results of my experiments. Can I use this blocking system in cell culture before addition of the external biotin or you have another suggestion. Thanks

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Answer

We tested this product either with fixed cells or with tissue sections in immunocytochemistry or Immunohistochemistry applications, living cells unfortunately were never tested. There are two main things to consider while using this product with living cells:
- Sodium Azide - diluting the reagent before use, should help minimizing its toxic effect however we would recommend checking in literature the minimum amount safer for cells viability. http://ria.ua.pt/bitstream/10773/4210/1/393.pdf
- Secondly, we will not be able to confirm whether this solution will permeate into cells so you may need to test this yourselves.
I am sorry we have no tested data to confirm that the product is suitable as per your requirement.

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Question

Quisiera hacer una pregunta acerca del complejo Avidin Biotin Abcam, la referencia es ab3387, mi pregunta es acerca de la caducidad, no he sabido ver límite de utilización ni en el producto en sí, ni en la página web Abcam.

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Answer

We do not have set expiration dates for our products. Most antibodies are stable and can last for anywhere from a few months to several years if stored properly, so we strongly recommend that you follow the storage instructions on the datasheet for the antibody you purchased. These conditions will vary among our antibodies, therefore, it is important to verify the storage conditions for each of our products when you receive them. We guarantee all of our products to work for 6 months from the date of purchase when stored correctly. As a guide, this product (ab3387) should last at least 1 year if stored correctly at 4 ºC.

For more information on antibody storage and stability, please visit our Antibody Storage Guide on our Protocols and Troubleshooting Tips page (https://www.abcam.com/index.html?pageconfig=popular_protocols).

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Question

I forgot to mention that we do a serum block before the primary antibody is applied.  We have an avidin-biotin blocking kit that we have tried with no effect on our results.  In our experience, we only need to use the avidin-biotin block in conjunction of a tyramide signal amplification (TSA) where there is too much background.  Unfortunately, too much signal isn't our issue, but too little.

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Answer

Thank you for your reply.

I would suggest changing your antigen retrieval method next. While you have used sodium citrate pH 6 and Tris/EDTA pH 10 I would recommend the following procedure based on a paper from Jiao et al which I will include.

In a water bath heat your samples in 10mM sodium citrate pH 9 at 80C for 30 minutes.

This procedure was created as en effective method for performing antigen retrieval for IHC0Fr but has been found equally effective in traditional FFPE IHC.

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

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Question

My lab has been having some IHC-P issues for a few weeks now and I was wondering if anyone on your team had an insight into our problem.   We use rhesus macaque brain (amongst other) tissue, and have been using the same samples for a few years. They were fixed in 10% formalin and put in paraffin wax blocks w/in 24 hours.     Our Protocol: We normally hydrate the tissue (Xylene, 100, 90, 70% EtOH, H2O), use a 10mM Sodium Citrate ph 6.0 with 1% Tween antigen retrieval 30min at 98C and 20min at room temp to cool. We then use a primary, biotinylated secondary, ABC-AP, and fast red equivalent (organic, not alcohol soluble), then we counterstain, dehydrate (70, 90, 100% EtOH, Xylene) and mount.   The problem we're having is that we are getting a severely reduced signal.  For our markers that are rare, we were getting little to no signal, while strong markers such as GFAP and MAP2 were there, but were much lighter.  We initially thought it was the chromogen, so we tried a newer ABC-AP, along with a newer substrate. We also tried Streptavidin-HRP with DAB, and avidin bound fluorophores which cut out the enzymes all together and just bound directly to the biotinylated 2ndary antibodies.  They all had reduced signal.   Our next thought was our antigen retrieval. Perhaps it wasn't unmasking enough. We tried our 10mM Sodium citrate pH6.0 with tween. We varied the tween from 0.01% all the way up to 2%.  We did see a gradient of signal stain, but it was still reduced overall.  We also tried a 10mM Tris 10mM EDTA pH9.0 with 0.1% tween in addition to a bought high and low pH buffer. None of these seemed to help.   We've tried several different combinations of primary and secondary antibodies, and the problem appears to be consistent.   I was hoping someone at your company would have some input/insight into our problem.  We are a regular user of your antibodies and I recently attended an IHC webinar that Abcam hosted.

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Answer

Thank you for contacting us.

I am sorry to hear of the troubles which you have been experiencing in IHC-P. In reviewing the protocol and troubleshooting provided I did not see that you block endogenous avidin or biotin. We highly recommend blocking of any endogenous biotin/avidin for any IHC-P ABC experiments, even with brain tissues. I would recommend using ab3387, Endogenous-Avidin-Biotin-Blocking-System for this. We would also highly recommend use of a serum/protein block such as ab128988 before use of any avidin/biotin blocking.

More information about our Endogenous Avidin Biotin Blocking system may be found at: https://www.abcam.com/Endogenous-Avidin-Biotin-Blocking-System-ab3387.html

Our Protein block is found at:
https://www.abcam.com/Protein-Block-ab128988.html

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



Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Question

The attached MSDS does not include the 1st page.  Please send me a complete MSDS for product number ab3387.  

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Answer

Thank you for contacting us regarding this matter.

We have updated the MSDS and have fixed this issue. We would like to thank you for bringing this to our attention. I gave included the updated MSDS here but it is available on the webpage for this product as well.

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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Question

Thanks for the reply.

We never did in our laboratory this protocol with Egg white and milk. Could you explain in more detail how and in what part of the procedure should I enter them.

I'm very pleased with the attention and speed that Abcam that serves customers.

Best regards,

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Answer

Thank you for your inquiry. My colleague is not in the office today and I am happy to answer your question instead of her. Please find below a protocol for blocking biotin with egg white:
1- Wash twice in TBS 0.05M pH7.5, to which 0.01% Tween 20 has been added (TBS-T).
2- Briefly blot the slides without letting them dry and then Apply egg white in PBS-BSA-NaN3 as a blocking agent (one egg white in 100 ml PBS + NaN3) (Miller RT et al, Appl Immunohistochemistry 5(1): 63-66, 1997). Incubate for 10 min. minimum. 3- Wash once in water.
4- Incubate with skim milk 5% in TBS-T for 10-30 min. (Miller RT et al, Appl. Immunohist & Molecular Morphology, 71(1): 63-65, 1999)
NOTE: skim milk may contain weak proteases which will be useful to further unmask antigens, but may reduce immunostaining by acting on primary Abs.
5- Wash once in TBS-T (residual milk can contribute to blocking).
This biotin blocking step can be added to the IHC protocol anywhere between the antigen retrieval and the immunostaining. I suggest to add this step to the general blocking step with serum.
Since you do not have any experience with egg white as blocking agent, I can recommend to use our biotin blocking kit ab3387:
https://www.abcam.com/index.html?datasheet=3387 https://www.abcam.com/index.html?datasheet=3387.
This will be easier to use and will also require less optimisation.
I hope this information was helpful and wish you good luck with your experiments.

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Question

I made more tests and still gives unexpected results, osteoblasts were labeled and not all osteoclasts were labeled.

I tried two new forms of antigen retrieval:

1- used skimmed milk to block the endogenous biotin

2- used bovine serum albun (BSA) 20% (the twice of the original protocol)

I'm sending in attached the pictures of my tests to you analyse my mistake (if possible).



Responding you questions...

1- I decalcificate my samples.

2- I tried use milk to block the endogenous biotin, but if the osteoblasts labeled for endogenous biotine the negative control would not going label too?

3- I did antigen retrieval using 10 mM citrate buffer (pH 6.0) in microwave



I don't know what else to try, because I´ve used almost half of antibody in test and still didn't work.



Looking forward to your answer.

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Answer

Thank you for contacting us.
Please be aware that the biotin problem is still not solved by using milk or BSA: those two will block the non-specific protein binding sites, but not the endogenous biotin. You can block the endogenous biotin by using either a ready-to-use avidin solution like our ab3387:
https://www.abcam.com/index.html?datasheet=3387 https://www.abcam.com/index.html?datasheet=3387.
Another, more simple method would be using egg white followed by skim milk. Egg white contains avidin, and skim milk is actually rich in biotin, and you need the free biotin to make the bound avidin unaccessible to your amplification system. Please rinse well after this step.
Regarding the antigen retrieval, I fear 10 min are not enough to crack the methylen bridges formed by the fixation agent. Please increase the time to a min of 20 min. so that the antibody can bind its target.
If you will see no improvement after those tips I will gladly provide a free of charge replacement, credit note, or refund (if the problem has been reported within 6 months of purchase).
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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Question

I am having trouble with Ki67 antibody (cat#ab15580). I used it in mouse tissues such as kidney, liver, testis, brain, cartilage etc. fixed in Bouin’s fixative and formaldehyde. The staining was with extremely high background. I tried 1:100 dilution at RT for 2hrs and also 1:200 O/N +4C0.

Besides the background staining, it stained cytoplasm of the cells! Here is my protocol: Retrieval: 10 mM sodium citrate buffer, pH6.0 in microwave, 5 min, twice. Cool down in the buffer for about 20 minutes. Wash in deionized water 3 times for 5 minutes each. Blocking for endogenous peroxidase with 3% H2O2 prepared in methanol for 20min, RT. Wash in deionized water 3 times for 5 minutes each. Blocking with UV block (Dako) for 7min, RT. Incubation with primary antibody Ki67 (ab15580) in antibody diluent (Thermo). Wash in PBS, 3 times, 5min each. Incubation with secondary antibody in PBS (vector, biotinylated anti rabbit; 1:500) for 30min, RT. Incubation with ABC substrate (Vector) for 30min, RT. Wash in PBS, 3 times, 5min each. DAB staining, wash, counterstaining and mounting with Cytoseal.

When I compared the Ki67 and PCNA (another proliferation marker) on the same slide, the difference was obvious. By the way, my negatives worked fine. I can send you some pictures as well. Thank you and I look forward to your reply.

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Answer

Thank you for contacting us.

Is the UV block from DAKO a protein block? We typically recommend performing a protein block using 10% normal serum from the host species of your secondary antibody. For example, if your secondary is goat anti-rabbit, we recommend blocking with 10% normal goat serum.

Additionally, when using ABC detection, endogenous avidin and biotin can sometimes cause diffuse, weak, cytoplasmic staining. To prevent this, you can use a system like ab3387.

I hope this will help to improve your results. If not, I will be happy to offer you a free of charge replacement, credit, or refund if you have purchased this antibody in the past six months. Please let me know your original order number and how you would like to proceed and I will be happy to assist you further.

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Question

Good morning, I'm currently using Abcam products to perform imunohistochemistry on paraffinon my research protocols and I have some doubts that I would likeAbcam tohelp me. I worked with Anti-Survivin antibody (ab469) as a primary antibody in a 1/500 dilution. I useda goat polyclonal secondary antibody (ab6720) as well as endogenous Avidin + Biotin Blocking System (ab3387) and DAB Substrate (ab94665)kit as a cromogen. With this experience Iwas able to get favorable results, however I would like to know how to get a more intense staining?I tried to optimize the dilution of primary antibody as well as the exposure time of DAB butcould not get significant changes. Could you give mean advise on how to get some more intense staining? Another question about the primary antibody Anti-Smac / Diablo (ab8114) is what dilution do you recomend using in IHC-P protocols? I read the datasheet where you say to use a concentration of 5ug/ml but I can't understand how to perform this concentration/ diluition. Best regards,

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Answer

Thank you for contacting us. I'm glad to hear you have been getting good staining with ab469. In order to help you obtain more intense staining would you mind filling in the form which I have attached to this email. It will allow me to more fully understand the protocol which you have been performing and any areas in which it may be worthwhile to optimise. Could you also answer the following questions: 1. which HRP conjugate have you been using (streptavidin-HRP? could you tell me the catalogue no. etc) 2. in what dilution have you been using the secondary antibody? ab8114 is supplied at a concentration of 1 mg/mL. The recommended working concentration of this antibody when using it for immunohistochemistry with paraffin embedded sections is 5 µg/ml. This equates to a dilution of 1/200. This is only a guideline and may need to be optimised for the staining which you are performing. I hope this information has been of help and look forward to your reply.

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Question

LOT NUMBER GR53220 ORDER NUMBER 975372 DESCRIPTION OF THE PROBLEM No staining SAMPLE mouse: lung, pancreas, CT26-Tumor, LPS-liver PRIMARY ANTIBODY 1:25 and 1:50 in PBS incubation overnight at roomtemperature DETECTION METHOD Steaptavidin-POD, 1:50, 10 min at RT POSITIVE AND NEGATIVE CONTROLS USED negative control: sections without primary antibody ANTIBODY STORAGE CONDITIONS -20°C FIXATION OF SAMPLE 4% Formalin ANTIGEN RETRIEVAL Citrat-buffer pH 6.0, microwave 600 Watt, 10 min PERMEABILIZATION STEP none BLOCKING CONDITIONS 3% H2O2 in Methanol for 10 min 3% Goatserum in PBS 30 min Endogenous Avidin + Biotin Blocking System (ab3387) SECONDARY ANTIBODY goat-anti-mouse biotin (ab98696, 1:100, 45 min at RT HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? dilution of primary antibody ADDITIONAL NOTES we used pancreas as postitive control because of the pictures in the datasheet

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Answer

Vielen Dank für Ihre Anfrage und dafür, dass Sie sich die Zeit genommen haben, unseren Fragebogen auszufüllen. Es tut mir leid, dass Sie Probleme mit unserem VEGF-Antikörper ab3109 hatten. Ich weiß, dass Sie schon viel Zeit in Ihre Experimente investiert haben und es ist enttäuschend, dass Sie bisher noch kein positives Ergebnis haben. Ich kann Sie darin bestätigen, dass die Pankreasschnitte auf jeden Fall positive Ergebnisse zeigen sollten. Laut Literatur eignet sich Lungengewebe auch als gute Positivkontrolle; Sie haben sich daher auf jeden Fall die richtigen Kontrollen ausgesucht. Bei der Durchsicht Ihres Protokolls ist mir aufgefallen, dass Sie als Sekundärantikörper ab98696 verwenden: Dies ist ein Antikörper gegen Maus-IgG2a, der noch weiteren Aufreinigungsschritten unterzogen wurde (pre-adsorbed), um eventuell vorhandene Kreuzreaktivitäten zu minimieren. Daher ist es sehr unwahrscheinlich, dass ab98696 den anti-VEGF- Antikörper ab3109 bindet, da dieser Primärantikörper vom IgG1-Isotyp (mit der kappa light chain) ist. Ich möchte Ihnen daher empfehlen, einen anderen Sekundärantikörper zu verwenden (zum Beispiel ab98691 Click here (or use the following: https://www.abcam.com/index.html?datasheet=98691).). Bei einer Inkubation ueber Nacht würden wir zwar eine Inkubationstemperatur von 4ºC empfehlen, da dies zu einem spezifischeren Signal führt. Abgesehen davon sieht Ihr Protokoll aber sehr gut aus und sollte positive Ergebnisse mit ab3109 und Ihren Proben bringen. Bitte lassen Sie mich wissen, ob ich Ihnen helfen konnte und zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

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