Anti-beta Actin antibody(AB8227)

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.

All lanes:

Western blot - Anti-beta Actin antibody (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 - Goat Anti-Rabbit IgG H&L (HRP) (ab205718) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 36 kDa

Observed band size: 42 kDa

Exposure time: 30s

All lanes:

Western blot - Anti-beta Actin antibody (ab8227) at 1 µg/mL

All lanes:

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

Secondary

Lanes 1 - 2:

Western blot - Donkey Anti-Rabbit IgG H&L (Alkaline Phosphatase) (ab97061) at 1/5000 dilution

Lanes 3 - 4:

Western blot - Donkey Anti-Rabbit IgG H&L (Alkaline Phosphatase) (ab97061) at 1/20000 dilution

Lanes 5 - 6:

Western blot - Donkey Anti-Rabbit IgG H&L (Alkaline Phosphatase) (ab97061) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 36 kDa

Exposure time: 3min

IHC image of beta actin staining in a section of formalin-fixed paraffin-embedded normal human colon*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was then incubated with ab8227, 1/1000 dilution, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody (ab6720, 1/1000 dilution) was used to detect the primary, and visualized using an HRP conjugated ABC system. Streptavidin HRP was used, ab7403 at a 1/10000 dilution. DAB was used as the chromogen (ab103723), diluted 1/100 and incubated for 10min at room temperature. The section was then 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:

Western blot - Anti-beta Actin antibody (ab8227) at 1/1000 dilution

Lanes 1 and 6:

HeLa nuclear lysate at 20 µg

Lanes 2 and 7:

HeLa whole cell lysate at 20 µg

Lanes 3 and 8:

A431 cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Lanes 10 and 5:

HEK 293 cell lysate at 20 µg

Lane 9:

Jurkate cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

Predicted band size: 41 kDa

Observed band size: 41.7 kDa

Exposure time: 5s

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.

All lanes:

Western blot - Anti-beta Actin antibody (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

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 5% Milk before being incubated with ab8227 overnight at 4°C. Antibody binding was detected using Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) (ab175781) secondary antibody at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

All lanes:

Western blot - Anti-beta Actin antibody (ab8227) at 1/1000 dilution

Lane 1:

A431 (human epidermoid carcinoma cell line) Whole Cell Lysate at 20 µg

Lane 2:

HEK-293 (human epithelial cell line from embryonic kidney) Whole Cell Lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryo fibroblast cell line) Whole Cell Lysate at 20 µg

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma cell line) Whole Cell Lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) (ab175781) at 1/10000 dilution

Predicted band size: 41 kDa

Observed band size: 42 kDa