IHC image of Erk1 (pT202/pY204) + Erk2 (pT185/pY187) staining in Human breast Adenocarcinoma formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ 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 20 mins. The section was then incubated with ab138482, 5µg/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.

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

Please note - This antibody detects all 4 modifications (T185 + Y187 in ERK2 and T202 + Y204 in ERK1) by Elisa, however we only detect 2 modifications (T185 in ERK2 and T202 in ERK1) by western blot.

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 ab138482 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406

Western blot analysis of Rat ERK1 + ERK2 (phospho T185 + Y187 + T202 + Y204) using ab138482 at 1/1000.

Lane 1 - Bacterial expressed ERK2 negative control.

Lane 2 - Bacterial expressed ERK2 with MEK1 R4F, positive control.

Lane 3 - PP2A treated phosphorylated ERK2.

Lane 4 - PTP1 U323 treated phosphorylated ERK2. M, Molecular weight marker.

ERK2 is phosphorylated at T183 and Y185. This antibody detects the dual phospho, and the pT183 forms. After treating the pT183 pY185 ERK2 with the serine/threonine phosphatase PP2A the antibody does not detect it. After treating the pT183 pY185 ERK2 with the tyrosine phosphatase PTPi U323 the antibody does detect it.

A IRDye^® 680-conjugated donkey anti-rabbit IgG (H+L) polyclonal (1/10000) was used as the secondary antibody.

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ab138482 was tested using an Indirect ELISA approach. The wells were coated with peptide (1µg/ml at 100µl/well) overnight at 4°C, followed by a 5% BSA blocking step for 1 hour at room temperature. The primary Ab was then added at a dilution range of 1- 0.00025µg/ml (100µl/well) for 1hr at room temperature. A HRP-conjugated anti-rabbit IgG (heavy and light chain) was used as a secondary antibody at 1:20,000 dilution for 1hr at room temperature.