IHC image of gp100 staining in a section of formalin-fixed paraffin-embedded Human Skin Melanoma*, performed on a Leica BOND^TM. 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 ab205469, 1/50 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

All lanes : HRP Anti-Melanoma gp100 antibody [EP4863(2)] (ab205469) at 1/1000 dilution

Lane 1 : B16-F0 (Mouse) Whole Cell Lysate
Lane 2 : Melanoma (Human) Whole Cell Lysate - tumor tissue

Lysates/proteins at 10 µg per lane.

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 70 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?


Exposure time: 8 minutes

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 3% milk before being incubated with ab205469 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.