Donkey Anti-Rabbit IgG H&L (HRP)

6 product images

• 

  Western blot - Donkey Anti-Rabbit IgG H&L (HRP) (ab205722)

  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 Anti-beta Actin antibody - Loading Control ab8227 overnight at 4°C. Antibody binding was detected using ab205722, and visualised using ECL development solution ECL Substrate Kit (High Sensitivity) ab133406.

  All lanes: Western blot - Anti-beta Actin antibody - Loading Control (Anti-beta Actin antibody - Loading Control ab8227) at 1 µg/mL

  Lane 1: Liver (Human) Tissue Lysate at 10 µg

  Lane 2: Liver (Mouse) Tissue Lysate at 10 µg

  Lane 3: Liver (Rat) Tissue Lysate at 10 µg

  Lane 4: HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

  Lane 5: NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

  Lane 6: PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µg

  Secondary

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

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 36 kDa

  Observed band size: 42 kDa

  Exposure time: 10s

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Donkey Anti-Rabbit IgG H&L (HRP) (ab205722)

  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 Anti-Histone H4 antibody [EPR16599] - ChIP Grade 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

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Donkey Anti-Rabbit IgG H&L (HRP) (ab205722)

  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 Anti-beta Tubulin antibody - Loading Control 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

• 

  Western blot - Donkey Anti-Rabbit IgG H&L (HRP) (ab205722)

  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 Anti-STAT3 antibody [EPR787Y] ab68153 overnight at 4°C. Antibody binding was detected using ab205722, and visualised using ECL development solution ECL Substrate Kit (High Sensitivity) ab133406.

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

  Lane 1: A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

  Lane 2: Heart (Mouse) Tissue Lysate at 10 µg

  Lane 3: Heart (Rat) Tissue Lysate at 10 µg

  Secondary

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

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 36 kDa

  Observed band size: 88 kDa

  Exposure time: 20min

• 

  Western blot - Donkey Anti-Rabbit IgG H&L (HRP) (ab205722)

  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 ECL Substrate Kit (High Sensitivity) ab133406.

  All lanes: No Primary Antibody

  Lane 1: Liver (Human) Tissue Lysate at 10 µg

  Lane 2: Liver (Mouse) Tissue Lysate at 10 µg

  Lane 3: Liver (Rat) Tissue Lysate at 10 µg

  Lane 4: HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

  Lane 5: NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

  Lane 6: PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µg

  Secondary

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

  Performed under reducing conditions.

  Exposure time: 10s

• 

  ELISA - Donkey Anti-Rabbit IgG H&L (HRP) (ab205722)

  Cross-reactivity of the polyclonal secondary antibody Donkey Anti-Rabbit IgG H&L 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. Donkey Anti-Rabbit IgG H&L 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) (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, Donkey Anti-Rabbit IgG H&L 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 (Donkey Anti-Rabbit IgG H&L ab182020).