Anti-KAP1 抗体 [20C1] (ab22553)

Western blot analysis was performed on nuclear enriched extracts (30 µg lysate) of cells treated with Hydrogen peroxide (100 µm Hydrogen peroxide for 10 min) or untreated. The blots were probed with ab22553 at 1 µg/mL and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate. A 110 kDa band corresponding to KAP1 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex^® NuPAGE^® 4-12 % Bis-Tris gel, XCell SureLock™ Electrophoresis System and Novex^® Sharp Pre-Stained Protein Standard. Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot^® 2 Dry Blotting System. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate.

IHC image of KAP1 staining in Human normal spleen formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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 ab22553, 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.