Overview

  • Product name
    Donkey Anti-Rabbit IgG H&L (HRP)
    See all IgG secondary antibodies
  • Host species
    Donkey
  • Target species
    Rabbit
  • Specificity
    The antibody used for conjugation reacts with rabbit immunoglobulins of all classes. Cross-reactions as determined by ELISA for the unconjugated antibody (ab182020): Human IgG, mouse IgG, rat IgG, goat IgG and chicken IgY, less than 2%.
  • Tested applications
    Suitable for: IHC-P, IP, WB, ELISAmore details
  • Immunogen

    The details of the immunogen for this antibody are not available.

  • Conjugation
    HRP

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. Store In the Dark.
  • Storage buffer
    pH: 7.4
    Preservative: 0.1% Proclin
    Constituents: PBS, 1% BSA, 30% Glycerol
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Purification notes
    This antibody was isolated by affinity chromatography using antigen coupled to agarose beads and conjugated to Horse Radish Peroxidase (HRP).
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab205722 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 1/2000 - 1/20000.
IP Use at an assay dependent concentration.
WB 1/2000 - 1/50000.
ELISA Use at an assay dependent concentration.

Images

  • All lanes : Anti-beta Actin antibody (ab8227) at 1 µg/ml

    Lane 1 : Liver (Human) Tissue Lysate
    Lane 2 : Liver (Mouse) Tissue Lysate
    Lane 3 : Liver (Rat) Tissue Lysate
    Lane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 5 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
    Lane 6 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Donkey Anti-Rabbit IgG H&L (HRP) (ab205722) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Observed band size: 42 kDa
    why is the actual band size different from the predicted?


    Exposure time: 10 seconds


    This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab8227 overnight at 4°C. Antibody binding was detected using ab205722, and visualised using ECL development solution ab133406.

  • IHC image of Histone H4 staining in a section of formalin-fixed paraffin-embedded normal human colon tissue*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins, and incubated overnight at +4°C with ab177840 at 1ug/ml. An HRP-conjugated secondary (Ab205722, 1/10000 dilution) was used to detect the primary for 1hr at room temperature. DAB was used as the chromogen (ab103723), diluted 1/100 and incubated for 10min at room temperature. The section was counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  • All lanes : Anti-beta Actin antibody (ab8227) at 1 µg/ml

    Lane 1 : Liver (Mouse) Tissue Lysate
    Lane 2 : Liver (Rat) Tissue Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : ab205722 (Left Image) at 1/10,000 and a competitor secondary (Right Image) at 1/10,000. Notice the decreased signal of the competitor product.

    Performed under reducing conditions.

    Observed band size: 42 kDa why is the actual band size different from the predicted?


    Exposure time: 10 seconds


    This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab8227 overnight at 4°C. Antibody binding was detected using ab205722 (Left Image) and a competitor secondary (Right Image), and visualised using ECL development solution ab133406.

  • IHC image of beta tubulin staining in a section of formalin-fixed paraffin-embedded normal human colon tissue*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins, and incubated overnight at +4°C with ab6046 at 5ug/ml. An HRP-conjugated secondary (Ab205722, 1/10000 dilution) was used to detect the primary for 1hr at room temperature. DAB was used as the chromogen (ab103723), diluted 1/100 and incubated for 10min at room temperature. The section was counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  • All lanes : No Primary Antibody

    Lane 1 : Liver (Mouse) Tissue Lysate
    Lane 2 : Liver (Rat) Tissue Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : ab205722 (Left Image) 1/2000 and a competitor secondary (Right Image) 1/2000. Notice the increased background of the competitor product.

    Performed under reducing conditions.

    Exposure time: 10 seconds


    This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was incubated overnight with 2% Bovine Serum Albumin at 4°C. Any non-specific background binding was assessed by incubating the membrane with ab205722 (Left Image) and a competitor secondary (Right Image), and visualised using ECL development solution ab133406.

  • All lanes : Anti-STAT3 antibody [EPR787Y] (ab68153) at 1/2000 dilution

    Lane 1 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : Heart (Mouse) Tissue Lysate
    Lane 3 : Heart (Rat) Tissue Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Donkey Anti-Rabbit IgG H&L (HRP) (ab205722) at 1/2000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Observed band size: 88 kDa why is the actual band size different from the predicted?


    Exposure time: 20 minutes


    This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab68153 overnight at 4°C. Antibody binding was detected using ab205722, and visualised using ECL development solution ab133406.

  • All lanes : No Primary Antibody

    Lane 1 : Liver (Human) Tissue Lysate
    Lane 2 : Liver (Mouse) Tissue Lysate
    Lane 3 : Liver (Rat) Tissue Lysate
    Lane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 5 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
    Lane 6 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Donkey Anti-Rabbit IgG H&L (HRP) (ab205722) at 1/2000 dilution

    Performed under reducing conditions.

    Exposure time: 10 seconds


    This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was incubated overnight with 2% Bovine Serum Albumin at 4°C. Any non-specific background binding was assessed by incubating the membrane with only the secondary antibody (ab205722), and visualised using ECL development solution ab133406.

  • Cross-reactivity of the polyclonal secondary antibody ab182020 was tested using a sandwich ELISA approach. The wells were coated with the indicated IgG standards at 1 µg/ml (50 µl/well) and incubated overnight at 4°C, followed by a 5% BSA blocking step for 2h at RT. ab182020 was then added starting at 1 µg/ml and gradually diluted 1/4 (50 µl/well), followed by incubation for 2h. For the detection Goat anti-Donkey IgG H&L (HRP) (ab6988) was used at 1/20,000 dilution (50 µl/well), followed by incubation for 1h at RT.

    For the batch tested, ab182020 showed a cross-reactivity below 2% towards human IgG, mouse IgG, rat IgG, goat IgG and chicken IgY.

    This data was developed using the unconjugated antibody (ab182020).

References

ab205722 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

Abreviews
Application
Western blot
Human Cell lysate - whole cell (Liver).
Loading amount: 5 µg.
Gel Running Conditions: Reduced Denaturing (10%).
Blocking step: 5%BSA/1%OvaAlb/PBS as blocking agent for 3 hours at room temperature.
Primary antibody: CRY2 (ab38872) diluted 1:1000 in 1%BSA/0.2%OvaAlb/PBS 14 hours at 4°C.
Secondary antibody: ab205722 - Donkey Anti-Rabbit IgG H&L (HRP) diluted 1:5000 in TBST.
ECL prime: 5 seconds.
Predicted size: 67kDa.

Abcam user community

Verified customer

Submitted Aug 12 2016

Application
Western blot
Excellent specificity.
Good sensitivity.
WB conditions:
- Cell lisates: 20 μg protein
- non-reducing
- Primary antibody: Rabbit anti-LaminB1, ab133741. Abcam. Dilution 1:5000
- Secondary antibody: 1:6000
- Developer: ECL Prime, Amersham, GE Helathcare Life Sciences, Product code RPN2232
- Exposure time: 1 second, 2nd film out 4

Abcam user community

Verified customer

Submitted Feb 01 2016

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