Overview

  • Product name
    Goat Anti-Rabbit IgG H&L (HRP)
    See all IgG secondary antibodies
  • Host species
    Goat
  • Target species
    Rabbit
  • Tested applications
    Suitable for: IHC-P, WB, ELISA, Immunomicroscopy, Dot blot, ICC, IHC-Frmore details
  • Immunogen

    Rabbit IgG, whole molecule

  • Conjugation
    HRP

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at -20°C. Avoid freeze / thaw cycle. Please see notes section.
  • Storage buffer
    pH: 7.4
    Preservative: 0.1% Proclin
    Constituents: 1% BSA, 30% Glycerol, PBS
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Purification notes
    This product was prepared from monospecific antiserum by immunoaffinity chromatography using Rabbit IgG coupled to agarose beads.
  • Conjugation notes
    Horseradish Peroxidase (HRP)
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • General notes

    HRP conjugated anti-rabbit secondary antibody optimized for western blot and immunohistochemistry. Some customers reported seeing brown precipitates in the vials. The brown precipitates are very common with HRP conjugated antibodies; we suggest vortexing the vial and using this antibody as normal. Our customer’s feedback says the antibody worked great. If in case the antibody fails to give results then please contact our Scientific Support team for assistance.

    For extended storage aliquot contents and freeze at -20° C or below. Centrifuge product if not completely clear after standing at room temperature.  This product is stable for several weeks at 4° C as an undiluted liquid.  Dilute only prior to immediate use.

     

  • Research areas

Applications

Our Abpromise guarantee covers the use of ab6721 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 1/1000.
WB 1/2000 - 1/20000.

Suggested working dilution of 1/3000 (see PMID: 17222046). In addition, found to work at 1/20000 (see PMID: 16936283).

Working dilutions are highlighted in the table below. Please note that the antibody can be diluted to 1:48,000 to 1:207,000 in many instances.

ELISA 1/120000.
Immunomicroscopy Use at an assay dependent concentration.
Dot blot Use at an assay dependent concentration.
ICC 1/1000 - 1/5000.
IHC-Fr 1/1000.

Images

  • Anti-phospho-tau immunostaining in Dryocopus lineatus.

    Tau-positivity in the midbrain (Panel A (shown) and B) and the corpus callosum (C) of the Dryocopus lineatus brain. The axonal tract staining demonstrates a thread-like pattern, similar to that seen with Gallyas sliver staining. Occasional intracellular tau-accumulations were identified within neurons (D).

    For full method, please see paper.

  • All lanes : Anti-NEK7 antibody (ab80948) at 1/2000 dilution

    All lanes : Mouse brain tissue cytoplasmic lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Exposure time: 10 seconds


    Blocked with 5% non-fat milk for 1 hour at 18°C

  • ab3580 staining glucocorticoid receptor in mouse epididymis tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections).

    Tissue was fixed with Bouin's solution and blocked with 1.5% serum for 30 minutes at 25°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/1000) for 14 hours at 4°C. An HRP-conjugated goat anti-rabbit IgG H&L (ab6721) (1/200) was used as the secondary antibody.

    See Abreview

  • Immunohistochemical analysis of PFA-fixed paraffin-embedded rat cardiac tissue sections, labeling Conexin 43 with ab117843 at a dilution of 1/500 incubated for 12 hours at 4°C in 1% BSA in TBS. Antigen retrival was via Tris-EDTA pH 9.0 (heat mediated). Blocking was 3% BSA incubated for 1 hour at 37°C. The secondary was ab6721 at 1/500.

    See Abreview

  • ab6721 was used at dilution 1/100 with the primary antibody ab11370 in IHC-P. See the review on ab11370.

  • ab6721 was used at dilution 1/100 with the primary antibody ab35604 in IHC-Fr. See the review on ab35604.

References

This product has been referenced in:
  • Wu Q  et al. MicroRNA-92b promotes cell proliferation and invasion in osteosarcoma by directly targeting Dickkopf-related protein 3. Exp Ther Med 15:173-181 (2018). Read more (PubMed: 29250147) »
  • Hinderer S  et al. Surface functionalization of electrospun scaffolds using recombinant human decorin attracts circulating endothelial progenitor cells. Sci Rep 8:110 (2018). Read more (PubMed: 29311692) »
See all 949 Publications for this product

Customer reviews and Q&As

1-10 of 19 Abreviews or Q&A

Application
Western blot
The dilution used for this secondary antibody was 1/20000 and we obtained intesnse specific bands even at very short incubation times. It works very well with phosphorylated proteins which sometimes are harder to detect (in these cases we use 1/10000 dilution or 1/5000).

Abcam user community

Verified customer

Submitted Oct 03 2018

ab6721

Excellent
Abreviews
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
This HRP-conjugated antibody works very well.

Dr. Shyambabu Chaurasiya

Verified customer

Submitted Jan 24 2018

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Very clear signal. The image is of a cross section of 3 metatarsal bones, primary antibody is Periostin (ab14041).

Haydee Gutierrez

Verified customer

Submitted Jan 09 2017

Abreviews
Application
Western blot
Secondary antibody (ab6721) was used at a dilution of 1:5000 in 5% BSA. Placental lysate was used. primary antibody was purchased for VEGFR1 (ab 32152) antibody.
gel: 10%, 200 V for 35 min.
transfer 17 V for 35 min. using Biorad TURBO
Blocking 5% BSA.

Vicky

Verified customer

Submitted Nov 07 2016

Application
Western blot
Experimental conditions:
Bone tissue lysate (tibia) extracted with RIPA buffer
10% SDS-PAGE
Blocking 1h with 5% milk in TBS-T at RT
Incubation with primary AB 1:1000 overnight at 4ºC
Incubation with secondary AB (ab6721 - Goat Anti-Rabbit IgG) 1:2000 for 1h at RT
Developed with ECL for 537s
Results
No nonspecific binding. No background. Clear bands.

Mr. Helder Fonseca

Verified customer

Submitted Oct 07 2014

Answer


In the case of ab6721, the storage condition has changed because we removed the glycerol bit from the antibody storage buffer. As a consequence, from now on we recommend to store the antibody at 4 degrees.
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).

Read More

Answer

The dilution factor on the datasheet only a recommendation for starting. The actual dilutions need to be optimized by the scientist since it depends on many factors such expression levels of the target in the samples, primary antibody (clonality, purification, specificity), signal amplification, detection system etc.

Read More

Answer

The main difference in the 3 secondary antibodies you mentioned are in the applications that they are tested and guaranteed to work in for 6 months as stated in our AbPromise guarantee, the method in which they were purified, and whether or not they are labeled as our "AbExcel" antibodies. Below I summarize these differences for you:

ab6721 (https://www.abcam.com/goat-polyclonal-secondary-antibody-to-rabbit-igg-hl-hrp-ab6721.html)
Tested applications: IHC-P, WB, ELISA, Immunomicroscopy, Dot Blot, ICC/IF, IHC-Fr
Purification method: IgG fraction
AbExcel: No

ab97051 (https://www.abcam.com/goat-polyclonal-secondary-antibody-to-rabbit-igg-hl-hrp-ab97051.html)
Tested applications: ICC, IHC-P, ELISA, WB
Purification method: Immunogen affinity purified
AbExcel: Yes (description of AbExcel: https://www.abcam.com/index.html?pageconfig=resource&rid=13534)

ab136636 (https://www.abcam.com/goat-polyclonal-secondary-antibody-to-rabbit-igg-hrp-ab136636.html)
Tested applications: ELISA, WB, IP
Purification method: Unlisted
AbExcel: No

Read More

Answer

We are aware of this issue; the brown precipitates as observed are very common with HRP conjugated secondary antibodies so our lab is devising ways to get rid of these precipitates however results are still awaiting.

We and our customers have tested this antibody many times with no problem, so I would suggest using the antibody .

Please vortex the vials and then centrifuge it. It is true that if you take out precipitates, there will be small decrease in concentration however it will not be affecting the sensitivity of the antibody.

Read More

Answer

I can confirm that for the HRP conjugated antibodies the approach that I recommended is the correct one. Spining the vial down will prevent free HRP to bind to unspecifically to samples and therefore avoid increased background.

If the antibody is not purified and therefore also not conjugated then we would recommend gentle vortexing. This could be whole antiserum for example.
In this case the antibody does contain proteins and other solutes it would not be abnormal to see a little cloudiness or precipitation, especially after being stored at a low temperature and/or for a long period of time. Most of the time bringing the antibody up to room temperature, mixing it well and then diluting it to a working concentration will help incorporate any protein aggregates and precipitation.

Read More

1-10 of 19 Abreviews or Q&A

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