HRP Anti-alpha smooth muscle Actin antibody [1A4]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - HRP Anti-alpha smooth muscle Actin antibody [1A4] (AB203696)

IHC image of alpha smooth muscle Actin staining in a section of formalin-fixed paraffin-embedded normal human colon*, performed on a Leica BOND™. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab203696, 1/100 dilution, for 15 mins at room temperature. DAB was used as the chromogen. 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

• WB

Lab

Western blot - HRP Anti-alpha smooth muscle Actin antibody [1A4] (AB203696)

ab203696 was shown to react with alpha smooth muscle Actin (HRP) in wild-type HeLa cells in western blot. Loss of signal was observed when ACTA2 knockout cell line ab264014 (knockout cell lysate ab264499) was used. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab203696 overnight at 4°C at a 1 in 5000 dilution and ab184095 (Mouse Anti-GAPDH antibody [mAbcam 9484] - Alexa Fluor^® 680) at a 1 in 1000 dilution. Blots were developed with Optiblot ECL reagent (ab133456) and imaged.

All lanes:

Western blot - HRP Anti-alpha smooth muscle Actin antibody [1A4] (ab203696) at 1/5000 dilution

Lane 1:

Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

ACTA2 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Predicted band size: 42 kDa

Observed band size: 42 kDa

false

Exposure time: 20s

• WB

Lab

Western blot - HRP Anti-alpha smooth muscle Actin antibody [1A4] (AB203696)

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 ab203696 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.

All lanes:

Western blot - HRP Anti-alpha smooth muscle Actin antibody [1A4] (ab203696) at 1/5000 dilution

Lane 1:

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

Lane 2:

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

Lane 3:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg

Predicted band size: 42 kDa

Observed band size: 42 kDa

true

Exposure time: 1min