Biotinylated Goat Anti-Rabbit IgG (H+L) (Ready to use) (ab64256)

Overview

  • Product name

    Biotinylated Goat Anti-Rabbit IgG (H+L) (Ready to use)
    See all IgG reagents
  • Tested applications

    Suitable for: IHC-Pmore details
  • General notes

    Staining protocol
    1. Deparaffinize and rehydrate formalin-fixed paraffin-embedded tissue section.
    2. Add appropriate hydrogen peroxide blocking solution 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 appropriate protein blocking solution and incubate for 5 minutes at room temperature to block non specific 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 Ready to use Biotinylated Goat anti Rabbit IgG (ab64256) solution and incubate for 10-15 minutes at room temperature. Wash 4 times in buffer.
    7. Apply enzyme labelled 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 Haematoxylin 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 ab64256 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-P Use at an assay dependent dilution.

Images

  • Ab64256 was used with the primary antibody ab3517 in IHC-P. See Abreview on ab3517.

References

This product has been referenced in:

  • Zhi X  et al. Adrenergic modulation of AMPK-dependent autophagy by chronic stress enhances cell proliferation and survival in gastric cancer. Int J Oncol 54:1625-1638 (2019). Read more (PubMed: 30896863) »
  • Ampofo E  et al. Maslinic acid alleviates ischemia/reperfusion-induced inflammation by downregulation of NF?B-mediated adhesion molecule expression. Sci Rep 9:6119 (2019). Read more (PubMed: 30992483) »
See all 28 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Answer

Thank you for contacting us about the Biotinylated Goat Anti Rabbit IgG antibody, ab64256.

The ratio of biotin to antibody is estimated to be 6 - 8. The concentration of the antibody is 5ug/ml.

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

Thank you for your inquiry. ab64257 is indeed suitable for the detection of Mouse and Rabbit IgG. It can be used as a secondary antibody when used with rabbit or mouse primary antibodies. About ab64256 and ab97044, all of the information we have for each of our products is listed on the online datasheet for your convenience. These are updated as soon as any new information is brought to our attention. Please do not hesitate to contact us again if you need further assistance.

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