Recombinant HRP Anti-Glutathione Peroxidase 1 antibody [EPR3312] (ab197034)

ab197034 was shown to recognize Glutathione Peroxidase 1 in wild-type HAP1 cells as signal was lost at the expected MW in GPX1 (Glutathione Peroxidase 1) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and GPX1 (Glutathione Peroxidase 1) knockout samples were subjected to SDS-PAGE. Ab197034 and ab184095 (Mouse monoclonal [mAbcam 9484] to GAPDH - Loading Control (Alexa Fluor^® 680) loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/1000 dilution respectively. The loading control was imaged using the Licor Odyssey CLx prior to blots being developed with ECL technique.

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 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 ab197034 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.

IHC image of Glutathione Peroxidase 1 staining in a section of formalin-fixed paraffin-embedded human normal 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 ab197034, at a dilution of 1/50, 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