Biotinylated Goat anti-Mouse IgG (H+L) (Ready to Use) (ab64255)

Overview

  • Product name

    Biotinylated Goat anti-Mouse IgG (H+L) (Ready to Use)
    See all IgG reagents
  • Tested applications

    Suitable for: Sandwich ELISA, IHC-Pmore details
  • General notes

    STAINING PROTOCOL:
    1. Deparaffinize and rehydrate formalin-fixed paraffin-embedded tissue section.
    2. Add enough drops of an approriate Hydrogen Peroxide Block to cover the sections. Incubate for 10 minutes. Wash 2 times in buffer.
    3. Perform appropriate pretreatment if required. Wash 3 times in buffer.
    4. Apply an appropriate Protein Block and incubate for 5 minutes at room temperature to block nonspecific background staining. Wash 1 time in buffer.
    5. Apply primary antibody and incubate according to manufacturer's protocol.
    6. Wash 4 times in buffer. Apply Biotinylated Goat anti Mouse IgG (H+L) solution and incubate for 10 minutes at room temperature. Wash 4 times in buffer.
    7. Apply enzyme labeled streptavidin and incubate according to manufacturer’s protocol.
    8. Rinse 4 times in buffer. Place slide in appropriate chromogenic substrate and incubate until desired reaction is achieved. Rinse 4 times in buffer.
    9. Add enough drops of Hematoxylin to cover the section. Incubate for 1 minute.
    10. Rinse 7-8 times in tap water. Add Mounting Medium to cover the section.


    This reagent is intended to be used in a labeled streptavidin-biotin immunoenzymatic antigen detection system. This technique involves the sequential incubation of the specimen with an unconjugated primary antibody specific to the target antigen, a biotinylated secondary antibody which reacts with the primary antibody, enzyme-labeled streptavidin, and substrate-chromogen.

Properties

Applications

Our Abpromise guarantee covers the use of ab64255 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
Sandwich ELISA Use at an assay dependent concentration. PubMed: 23754944
IHC-P Use at an assay dependent dilution.

Images

  • Human esophagus tissue section stained with biotinylated Goat anti Mouse IgG (H+L) (ab64255).

References

This product has been referenced in:

  • Blackwood CA Jagged1 is Essential for Radial Glial Maintenance in the Cortical Proliferative Zone. Neuroscience 413:230-238 (2019). Read more (PubMed: 31202705) »
  • Khodayari Bavil A & Kim J A capillary flow-driven microfluidic system for microparticle-labeled immunoassays. Analyst N/A:N/A (2018). Read more (PubMed: 29878004) »
See all 3 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Question
Answer

The ratio is approximately 6-8 biotins per antibody.

I hope this is helpful. Please contact us again if you have any further questions.

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Question
Answer

The concentration is always 7ug/mL for this product.
Please let me know if there is additional information I can provide.

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Answer

Thank you for your enquiry.

Yes you can use ab64255 for ELISA. Please note it comes pre-diluted and is meant to be used neat. Further dilution is possible but if signal intensity is low you could not increase concentration of the secondary.

I will look into the concentration of ab64255 and follow up with you as soon as I have more details.

I hope this is helpful. Pleasecontact me again if you have any further questions.

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Answer

Thank you for contacting us.

This is a ready to use product. You do not need to dilute it further.

However if you get high intensity of staining then you may dilute it 1/10, this way you can do more tests and antibody will last longer. Please be advised that this is just an advice based onmy own research experience.

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 us.

We do not currently offer double staining IHC kits. Your staining conditions are challenging as, of the two common HRP chromogens, only DAB is alcohol insoluable. While of the common AP chromagens, only BCIP/NBT is insoluable. This means that to do double labeling you will have to do a sequential stain first using one detection system and then with the other.

I have collected a list, with links, of the reagents which you would need for this type of staining.

1) After de-parrafinizing and re-hydrating the samples, perform an antigen retrieval step treating with Cirate buffer pH6.0,https://www.abcam.com/10x-Citrate-Buffer-pH-6-0-ab64214.html, in the microwave or pressure cooker. After performing the antigen retrieval rinse in cold tap water for 10 minutes.

2) Rinse slides in TBST,https://www.abcam.com/20x-TBS-T-with-Tween-20-ab64250.html.

3) Performing a blocking step using protein block,https://www.abcam.com/Protein-Block-ab64226.html.

4) Incubate sections with your first primary antibody, for clarity I will assume the CD44 antibody, raised in mouse.

5) Wash with TBST.

6) Block endogenous peroxidases using aHydrogen Peroxide Blocking Reagent,https://www.abcam.com/Hydrogen-Peroxide-Blocking-Reagent-ab94666.html.

7) Incubate your tissue with a biotinylated secondary antibody, in this instance anti-Mouse IgG,https://www.abcam.com/Biotinylated-Goat-anti-Mouse-IgG-H-L-Ready-to-Use-ab64255.html.

8) Wash with TBST.

9) Detect your first antibody usingStreptavidin Peroxidase (Ready to Use)https://www.abcam.com/Streptavidin-Peroxidase-Ready-to-Use-ab64269.html. Note that this does not containa serum or BSA solution as diluent. Serum may contain biotin therefore competing Streptavidin binding with biotinylated secondary antibody, therefore reducing binding activity.

10) Apply your chromogen, DABsubtrate,https://www.abcam.com/DAB-Substrate-Kit-ab64238.html.

11) Wash with TBST.

12)Performing a second blocking step using protein block,https://www.abcam.com/Protein-Block-ab64226.html.

13)Wash with TBST.

14)Incubate sections with the your second primary antibody, this time ALDH1, raised in rabbit.

15)Wash with TBST.

16)Incubate your tissue with a biotinylated secondary antibody, in this instance anti-Rabbit IgG,https://www.abcam.com/Biotinylated-Goat-Anti-Rabbit-IgG-H-L-Ready-to-use-ab64256.html

17)Wash with TBST.

18)Detect your first antibody usingStreptavidin Alkaline Phosphatase (Ready to Use)https://www.abcam.com/Streptavidin-Alkaline-Phosphatase-Ready-to-Use-ab64268.html.

19) Apply your second chromogenAlkaline Phosphatase chromogen (BCIP/NBT) - Ready to Use,https://www.abcam.com/Alkaline-Phosphatase-chromogen-BCIP-NBT-Ready-to-Use-ab7468.html.

18) Wash with TBST, counterstain if desired.


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

Vielen Dank für Ihren Anruf. ab64255 wurde über Affinitätschromatographie aus dem Antiserum aufgereinigt; der exakte Reinheitsgrade wurde leider nicht bestimmt. Der Puffer, in dem der Antikörper vorliegt, enthält BSA. Die Konzentration beträgt 7 µg/ml, und weil es sich um ein ready-to-use-Produkt handelt, bekommen Sie tatsächlich 125 ml ! Für Ihre Versuchsvorhaben könnte sich dieser Antikörper daher vielleicht nicht unbedingt eignen - weil sich 125 ml doch eher schlecht verarbeiten lassen. Gerne möchte ich Ihnen zwei Alternativen nennen, die in weniger Volumen erhältlich sind (ab97021 und ab97033). Beide Antikörper sind biotinyliert, ab97033 ist zusätzlich noch prae-absorbiert, d.h. er reagiert nicht mit den auf dem Datenblatt angegebenen Spezies (wie Huhn, Rind, Ziege etc): Click here (or use the following: https://www.abcam.com/index.html?datasheet=97021). Click here (or use the following: https://www.abcam.com/index.html?datasheet=97033). Ich hoffe, diese Informationen helfen Ihnen weiter. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

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