IHC image of Ubiquitin staining in mouse Alzheimer brain 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 ab33893, 1in500, 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.

All lanes : Anti-Ubiquitin antibody [EP296Y] (ab33893) at 1/1000 dilution

Lane 1 : MCF-7 Whole cell lysate
Lane 2 : MCF-7 Whole cell lysate + (50 uM 90 min)
Lane 3 : Mouse Brain

Lysates/proteins at 20 µg per lane.

Predicted band size: 8 kDa

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. ab33893 and ab8245 (loading control to GAPDH) were diluted 1/1000  and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging using the Licor Odyssey CLx.

Anti-Ubiquitin antibody [EP296Y] (ab33893) at 1/2000 dilution + Hela cell lysate.

Predicted band size: 8 kDa
Observed band size: 8.5 kDa why is the actual band size different from the predicted?

ab33893 at a 1:250 dilution staining Ubiquitin in human prostate adenocarcinoma using Immunohistochemistry, Paraffin Embedded Tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.