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 : Goat Anti-Rabbit IgG H&L (HRP) (ab205718) at 1/50000 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: 30 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 ab205718, 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 1/1000 dilution. An HRP-conjugated secondary (Ab205718, 1/20000 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 : ab205718 (Left Image) at 1/20,000 and a competitor secondary (Right Image) at 1/50,000. Notice the increased background 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: 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 then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab8227 overnight at 4°C. Antibody binding was detected using ab205718 (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 1/100 dilution. An HRP-conjugated secondary (Ab205718, 1/20000 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

IHC image of Ki67 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 ab15580 at 1/1000 dilution. An HRP-conjugated secondary (Ab205718, 1/20000 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

Cross-reactivity of the polyclonal secondary antibody ab182016 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. ab182016 was then added starting at 1 µg/ml and gradually diluted 1/4 (50 µl/well), followed by incubation for 2h. For the detection Donkey anti-Goat IgG H&L (HRP) (ab6885) was used at 1/10,000 dilution (50 µl/well), followed by incubation for 1h at RT.

For the batch tested, ab182016 showed a cross-reactivity of 5-7% towards Human IgG and below 2% towards Mouse IgG, Rat IgG and Chicken IgY.

This data was developed using the unconjugated antibody (ab182016).

Cross-reactivity of Goat anti-Rabbit IgG H&L (ab182016) and Goat anti-Rabbit IgG H&L obtained from two different vendors was tested using a sandwich ELISA approach. The wells were coated with the indicated IgG standards (Rabbit, Human, Mouse and Rat) at 1 µg/ml (50 µl/well) and incubated overnight at 4°C, followed by a 5% BSA blocking step for 2h at RT. Secondary antibodies were then added starting at 1 µg/ml and gradually diluted 1/4 (50 µl/well), followed by incubation for 2h. For the detection Donkey anti-Goat IgG H&L (HRP) (ab6885) was used at 1/10,000 dilution (50 µl/well), followed by incubation for 1h at RT. This data is from a representative dilution.

This data was developed using the unconjugated antibody (ab182016).