HRP Anti-Nestin antibody [rat-401] (ab196694) at 1/2000 dilution + MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 177 kDa
Observed band size: 170 kDa why is the actual band size different from the predicted?


Exposure time: 20 minutes

This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 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 ab196694 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.

IHC image of Nestin staining in a section of formalin-fixed paraffin-embedded rat 6 week brain (SVZ), 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 ab196694, 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.