IHC image of ERK2 staining in a section of formalin-fixed paraffin-embedded human pancreas carcinoma* 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 ab231085, 0.01ug/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

All lanes :

Lane 1 : Wild type HAP1 cell lysate
Lane 2 : ERK2 knockout HAP1 cell lysate
Lane 3 : NIH3T3 cell lysate
Lane 4 : PC12 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 41 kDa

ab231085 was shown to specifically react with ERK2 (MAPK1) in wild type HAP1 cells. No band was observed when ERK2 (MAPK1) knockout samples were used. Wild-type and ERK2 (MAPK1) knockout samples were subjected to SDS-PAGE. ab231085 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at a 1µg/ml concentration and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.