All lanes : Anti-Actin antibody [mAbGEa] (ab230169) at 1 µg/ml

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate
Lane 3 : PC12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 4 : Drosophila lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP conjugated Goat Anti-Mouse IgG (H+L) at 1/1500 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 42 kDa


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 55 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 ab230169 overnight at 4°C. Antibody binding was detected using a goat anti-mouse antibody conjugated to HRP, and visualised using ECL development solution ab133406.

IHC image of beta actin staining in a section of formalin-fixed paraffin-embedded normal human normal colon* performed on a Leica BOND^TM system using the standard protocol F. 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 ab230169, 1ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only 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