The antibody used for conjugation reacts with goat immunoglobulins of all classes.
Cross-reactions as determined by ELISA for the unconjugated antibody (ab182021): Human IgG, mouse IgG, rat IgG, rabbit IgG and chicken IgY, less than 2%.
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 ab8229 overnight at 4°C. Antibody binding was detected using ab205723, and visualised using ECL development solution ab133406.
Western blot - Donkey Anti-Goat IgG H&L (HRP) (ab205723)
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 Donkey Anti-Goat IgG H&L (HRP) (ab205723) at 1/2000 dilution
Performed under reducing conditions.
Exposure time : 5 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 (ab205723), and visualised using ECL development solution ab133406.
IHC image of alpha 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 ab7291 at 1ug/ml. Ab98800, goat anti-mouse IgG, was then added as a secondary bridging antibody, at 1/250 dilution for 1h.
An HRP-conjugated secondary (Ab205723, 1/2000 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
References for Donkey Anti-Goat IgG H&L (HRP) (ab205723)
This product has been referenced in:
Zhou Z et al. siRNA targeting YAP gene inhibits gastric carcinoma growth and tumor metastasis in SCID mice. Oncol Lett11:2806-2814 (2016).
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