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).